Collagen enhancing agent made from low acyl gellan gum, methods of making and methods of using the same

ABSTRACT

Described herein are materials and methods useful for the treatment of neurodegenerative disorders and bone disorders, as well as healing wounds, in a subject. Materials described herein include polysaccharide digestive products resulting from the enzymatic hydrolysis of low acyl gellan gum. Also described herein are pharmaceutical compositions comprising one or more of such materials, and methods for their preparation and use.

CROSS-REFERENCE TO RELATED CASES

This is a bypass continuation-in-part application of internationalapplication PCT/US16/17734, filed under the authority of the PatentCooperation Treaty on Feb. 12, 2016, published; which claims thepriority to U.S. Provisional Application No. 62/115,777, filed under 35U.S.C. § 111(b) on Feb. 13, 2015; to U.S. Provisional Application No.62/115,781, filed under 35 U.S.C. § 111(b) on Feb. 13, 2015; and to U.S.Provisional Application No. 62/115,786, filed under 35 U.S.C. § 111(b)on Feb. 13, 2015. The entire disclosures of all the aforementionedapplications are expressly incorporated herein by reference for allpurposes.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a progressive degenerative disorder of thebrain that begins with memory impairment and eventually progresses todementia, physical impairment, and death. Approximately 4.5 millionpeople in the United States suffer from AD, costing over $100 billionannually. During AD development, oxidative stress significantlyincreases inside of the brain, resulting in production of excessive freereactive radicals, such as reactive oxygen intermediates (ROI:superoxide anion and hydrogen peroxide) and 4-hydroxynonenal (4-HNE), alipid peroxide. Amyloid β peptide (Aβ₄₂) also increases oxidative stressby damaging the mitochondria, resulting in generation of free radicals.Cortical and hippocampal neurons exposed to Aβ₄₂ begin neurite atrophyand then undergo apoptosis.

Parkinson's disease affects nearly 1 million Americans and is the secondleading neurodegenerative disease in the United States. Parkinson'sdisease is a result of chronic progressive degeneration of neurons, thecause of which has not yet completely been clarified. While the primarycause of Parkinson's disease is not known, it is characterized by thepreferential loss of dopaminergic neurons in the substantia nigra andsubsequent loss of dopamine in the striatum. Loss of dopamine in thestriatum results in resting tremor, bradykinesia, rigidity, and posturalinstability.

Remedies are needed that not only influence the dopaminergictransmission and alleviate the symptoms of the Parkinson's disease inadvanced stages, but also reverse, prevent, or at least significantlydelay the dopaminergic neuron extinction in the early, to a great extentmotor-asymptomatic, Parkinson stages. However, the short half-life andblood-brain barrier (BBB) impermeability of protein-based neurotrophicfactors limit their clinical uses. Thus, there is a need for analternative neurotrophic agent that overcomes those shortcomings forbetter treatment of PD.

SUMMARY OF THE INVENTION

In a first aspect, there is provided herein a method of treating adegenerative neurological disorder comprising administering to a subjectin need of such treatment, an effective amount of low acyl gellan gum(LA-GAGR) or its cleavage product, thereby treating the degenerativeneurological disorder.

In certain embodiments, the degenerative neurological disorder isselected from the group consisting of Alzheimer's disease, Parkinson'sdisease, multiple sclerosis, amyotrophic lateral sclerosis, spinal cordinjury, and brain trauma. In certain embodiments, the subject is ahuman.

In certain embodiments, the effective amount of LA-GAGR or its cleavageproduct is administered in combination with a pharmaceuticallyacceptable carrier thereof.

In another aspect, there is provided herein a method of enhancingosteogenesis by adult mesenchymal stem cells comprising contacting oneor more adult mesenchymal stem cells with an effective amount of LA-GAGRor its cleavage product, thereby enhancing osteogenesis by adultmesenchymal stem cells.

In another aspect, there is provided herein a method of enhancingosteogenesis comprising administering to a subject in need thereof aneffective amount of LA-GAGR or its cleavage product, thereby enhancingosteogenesis. In certain embodiments, the subject has osteoarthritis,osteoporosis, a metabolic bone disorder, and/or a broken and/orfractured bone. In certain embodiments, the subject is human.

In certain embodiments, the LA-GAGR cleavage product is produced byenzymatic digestion of LA-GAGR by α(1→3)-glucosidase.

In certain embodiments, the enzymatic digestion of LA-GAGR byα(1→3)-glucosidase occurs at approximately 37° C. for about 48 hours.

In certain embodiments, the LA-GAGR cleavage product is midi-LA-GAGR,wherein midi-GAGR has a molecular weight of about 4,775 g/mol.

In certain embodiments, the enzymatic digestion of LA-GAGR byα(1→3)-glucosidase occurs at approximately 37° C. for ≥72 hours.

In certain embodiments, the LA-GAGR cleavage product is mini-GAGR,wherein mini-GAGR has a molecular weight of about 718 g/mol.

In certain embodiments, the enzymatic digestion of LA-GAGR byα(1→3)-glucosidase occurs in acetate buffer having a concentration ofabout 0.1 M.

In certain embodiments, the acetate buffer has a pH of about 5.

In certain embodiments, the acetate buffer comprises 1% salicin.

In another aspect, there is provided herein a composition comprising alow acyl gellan gum (LA-GAGR) cleavage product, wherein the LA-GAGRcleavage product is produced by enzymatic digestion of LA-GAGR byα(1→3)-glucosidase.

In certain embodiments, the composition further comprises apharmaceutically acceptable carrier for the LA-GAGR cleavage product.

In certain embodiments, the composition comprises 10-95% midi-GAGR,mini-GAGR, or a mixture thereof, a pharmaceutically acceptable carrierthereof, and wherein the composition is formulated for oraladministration.

In certain embodiments, the composition comprises 25-75% midi-GAGR,mini-GAGR, or a mixture thereof.

In certain embodiments, the pharmaceutical formulation comprises0.0001-20% by weight midi-GAGR, mini-GAGR or a mixture thereof, andwherein the composition is formulated for aerosol (inhalational)administration.

In certain embodiments, the composition comprises 1-10% by weightmidi-GAGR, mini-GAGR or a mixture thereof.

In another aspect, there is provided herein a method of treatingosteoporosis comprising administering to a subject in need thereof aneffective amount of midi-GAGR and/or mini-GAGR, thereby treatingosteoporosis. In certain embodiments, the subject is human.

In another aspect, provided herein is a method of enhancing woundhealing, the method comprising applying a composition comprising aneffective amount of LA-GAGR or its cleavage product(s) to a wound of asubject to enhance healing of the wound. In certain embodiments, whereinthe LA-GAGR cleavage product comprises midi-GAGR or mini-GAGR. Incertain embodiments, the LA-GAGR cleavage product is produced byenzymatic digestion of LA-GAGR by α(1→3)-glucosidase. In particularembodiments, the enzymatic digestion of LA-GAGR by α(1→3)-glucosidaseoccurs at approximately 37° C. for about 48 hours. In particularembodiments, the LA-GAGR cleavage product is midi-LA-GAGR, having amolecular weight of about 4,775 g/mol. In particular embodiments, theenzymatic digestion of LA-GAGR by α(1→3)-glucosidase occurs atapproximately 37° C. for ≥72 hours. In particular embodiments, theLA-GAGR cleavage product is mini-LA-GAGR, having a molecular weight ofabout 718 g/mol. In certain embodiments, the composition comprises agel. In particular embodiments, the gel composition comprises theLA-GAGR or LA-GAGR cleavage product bound to a gelling componentcomprising chitosan, alginate, and/or hyaluronic acid.

In another aspect, provided herein is a method of enhancing collagenproduction or glycosaminoglycan (GAG) production in cells, the methodcomprising administering an effective amount of a low acyl gellan gum(LA-GAGR) cleavage product to cells, and enhancing collagen productionor GAG production in the cells, wherein the LA-GAGR cleavage productcomprises midi-GAGR or mini-GAGR. In certain embodiments, the LA-GAGRcleavage product is produced by enzymatic digestion of LA-GAGR byα(1→3)-glucosidase. In particular embodiments, the enzymatic digestionof LA-GAGR by α(1→3)-glucosidase occurs at approximately 37° C. forabout 48 hours. In particular embodiments, the LA-GAGR cleavage productis midi-LA-GAGR, having a molecular weight of about 4,775 g/mol. Inparticular embodiments, the enzymatic digestion of LA-GAGR byα(1→3)-glucosidase occurs at approximately 37° C. for ≥72 hours. Inparticular embodiments, the LA-GAGR cleavage product is midi-LA-GAGR,having a molecular weight of about 718 g/mol. In certain embodiments,the LA-GAGR cleavage product is administered in a gel composition. Inparticular embodiments, the gel composition comprises the LA-GAGRcleavage product bound to a gelling component comprising chitosan,alginate, and/or hyaluronic acid. In certain embodiments, the cellscomprise cartilage cells.

In another aspect, provided herein is a method of enhancing blood vesselformation, the method comprising administering an effective amount ofLA-GAGR or its cleavage product to vein endothelial cells, and enhancingblood vessel formation in the vein endothelial cells, wherein theLA-GAGR cleavage product comprises midi-GAGR or mini-GAGR. In certainembodiments, the LA-GAGR cleavage product is produced by enzymaticdigestion of LA-GAGR by α(1→3)-glucosidase. In particular embodiments,the enzymatic digestion of LA-GAGR by α(1→3)-glucosidase occurs atapproximately 37° C. for about 48 hours. In particular embodiments, theLA-GAGR cleavage product is midi-LA-GAGR, having a molecular weight ofabout 4,775 g/mol. In particular embodiments, the enzymatic digestion ofLA-GAGR by α(1→3)-glucosidase occurs at approximately 37° C. for ≥72hours. In particular embodiments, the LA-GAGR cleavage product ismidi-LA-GAGR, having a molecular weight of about 718 g/mol. In certainembodiments, the LA-GAGR cleavage product is administered in a gelcomposition. In particular embodiments, the gel composition comprisesthe LA-GAGR cleavage product bound to a gelling component comprisingchitosan, alginate, and/or hyaluronic acid.

In another aspect, provided herein is a topical composition comprising apolysaccharide component comprising LA-GAGR, a cleavage product ofLA-GAGR, or combinations thereof; a gelling component bound to thepolysaccharide component, wherein the gelling component comprises one ormore of chitosan, alginate, or hyaluronic acid; and, optionally, one ormore pharmaceutically acceptable excipients, diluents, or adjuvants,wherein the topical composition is a gel. In certain embodiments, theLA-GAGR cleavage product is produced by enzymatic digestion of LA-GAGRby α(1→3)-glucosidase, and comprises midi-GAGR or midi-GAGR. In certainembodiments, the topical composition is configured for controlledrelease of the polysaccharide component.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: Low acyl gellan gum (LA-GAGR). Drawing of the chemicalstructure of LA-GAGR ([D-Glc(β1→4)D-GlcA(β1→4)D-Glc(β1→4)L-Rha(α1→3]n).Arrows point to either α- or β-glucosidic bonds. Enzymatic decompositionof LA-GAGR by α(1→3)-glucosidase results in cleavage products havingapproximate molecular weights of either 4,755 g/mol (48 hour digestion)or 718 g/mol (72 hour digestion).

FIG. 1B: Repeating tetrasaccharide unit of low acyl gellan gum. Low acylgellan gum consists of the repeating tetrasaccharide unit([D-Glc(β1→4)D-GlcA(β1→4)D-Glc(β1→4)L-Rha(α1→3)]_(n)).

FIG. 2: Rheometer measurement of low acyl gellan gum (LA-GAGR). LA-GAGRshows an average molecular weight of approximately 99,639 g/mol.

FIG. 3: Rheometer measurement of 24 hour LA-GAGR digestion product.After 24 hours of enzymatic hydrolysis, the average molecular weight ofLA-GAGR digestion product was reduced to approximately 30,245 g/mol. Thethree peaks shown indicate that α(1→3)-glucosidase breaks down LA-GAGRin multiple steps.

FIG. 4: Rheometer measurement of 48 hour LA-GAGR digestion product,midi-GAGR. 48 hour enzymatic digestion of LA-GAGR yielded a product ofapproximately 4,775 g/mol (midi-GAGR). The single narrow peak shownindicates that 48 hour digestion yielded a single product of anapproximate equivalent length (molecular weight).

FIG. 5: Rheometer measurement of 72 hour LA-GAGR digestion product,mini-GAGR. 72 hours of enzymatic digestion of LA-GAGR yielded a productof approximately 718 g/mol (mini-GAGR).

FIGS. 6A-6B: Midi-GAGR protects cortical neurons from the neurotoxicityof amyloid β peptide. Primary cortical neurons were treated with mock or1 μM of dextran (FIG. 6A), alginate, high acyl gellan gum, or midi-GAGR(FIG. 6B) for 6 h prior to the treatment with 10 μM amyloid β peptidefor 48 h. The viability of neurons was assessed using LIVE/DEADViability/Cytotoxicity Assay Kit. Dead cells appear red and live cellsappear green.

FIG. 6C: Midi-GAGR protects cortical neurons from the neurotoxicity ofamyloid β peptide. Depicted is a bar graph showing the percent of livecells (n>200 cells per condition×3, Mean±SEM).

FIG. 6D: Midi-GAGR protects cortical neurons from the neurotoxicity ofamyloid β peptide. Depicted is a flow chart summarizing the treatmentprotocol of primary cortical neurons used to generate FIGS. 6A-6C.

FIGS. 7A-7B: Midi-GAGR has neuroprotective effects on differentiatedPC12 cells and primary neurons. PC12 cells were differentiated in 100ng/ml NGF, treat with mock (FIG. 7A), midi-GAGR (FIG. 7B), dextran, oralginate for 6 h, and then with 6-OHDA for 24 h. Dead cells in red andlive cells in green.

FIG. 7C: Midi-GAGR has neuroprotective effects on differentiated PC12cells and primary neurons. Depicted is a bar graph showing the percentof dead cells (n>800 cells per condition×3, mean±SEM).

FIGS. 8A-8B: Midi-GAGR has neuroprotective effects on differentiatedPC12 cells and primary neurons. Primary cortical neurons were treatedwith mock (FIG. 8A) or 1 μM of midi-GAGR (FIG. 8B), dextran, alginate,or high acyl gellan gum for 6 h prior to the treatment with amyloid βpeptide for 24. Dead cells in read and live cells in green.

FIG. 8C: Midi-GAGR has neuroprotective effects on differentiated PC12cells and primary neurons. Depicted is a bar graph showing the percentof live cells (n>200 cells per condition×3, mean±SEM).

FIG. 9A: Midi-GAGR increases the levels of phosphorylated CREB in thenucleus. Differentiated PC12 cells were treated with mock or 1 μMmidi-GAGR, dextran, or alginate and stained with antibodies to α-tubulin(red) and pCREB (green) along with DAPI (blue).

FIG. 9B: Midi-GAGR increases the levels of phosphorylated CREB in thenucleus. Depicted is a bar graph showing the average intensities ofpCREB in the nucleus/cytoplasm (n=100 cells, mean±SEM).

FIGS. 10A-10B: Midi-GAGR increases the levels of phosphorylated CREB inthe nucleus. Mouse embryonic cortical neurons were treated with mock or1 μM dextran (FIG. 10A), alginate, high acyl gellan gum, or midi-GAGR(FIG. 10B) and processed for immunocytochemistry using antibodies toα-tubulin (red) and pCREB (green) along with DAPI (blue).

FIG. 10C: Midi-GAGR increases the levels of phosphorylated CREB in thenucleus. Depicted is a bar graph showing the average intensities ofpCREB in the nuclei (n=50 neurons, mean±SEM).

FIG. 10D: Midi-GAGR increases the levels of phosphorylated CREB in thenucleus. Depicted is a bar graph showing the average intensities ofpCREB in the nuclei.

FIGS. 11A-11D: Midi-GAGR has a neuritogenic activity. Embryonic corticalneurons were treated with mock (FIG. 11A) or 1 μM midi-GAGR (FIG. 11B)for 2 days and then with 25 μM 4-HNE (FIG. 11C: pretreated with mock;FIG. 11D: pretreated with midi-GAGR) for 1 day. Neurons were processedfor immunocytochemistry using antibody to synaptophysin (green) andphalloidin (actin: red) (scale bars=75 μm).

FIGS. 12A-12B: Midi-GAGR has a neuritogenic activity. Depicted is a linegraph showing the numbers of dendrite crossings (FIG. 12A), and a bargraph showing the number of synaptic clusters (per 15 μm) (FIG. 12B) incortical neurons treated with mock, 1 μM dextran or 1 μM midi-GAGR. Thenumbers of dendritic crossing and synaptic clusters were quantified byScholl ring analysis and Metamorph (n=30 neurons×2, mean±SEM,*=p<0.001).

FIG. 12C: Midi-GAGR has a neuritogenic activity. Depicted is a linegraph showing the numbers of crossings counted in neurons pretreatedwith mock, 1 μM dextran, 100 μM ascorbate, 1 μM high acyl gellan gum, or1 μM midi-GAGR, and then treated with 25 μM 4-HNE (n=30 neurons×2,mean±SEM, * (dark colored star)=p<0.01, * (light colored star)=p<0.001compared to mock).

FIG. 13A: Midi-GAGR has a neurite outgrowth enhancing effect in thepresence of 25 μM 4-HNE. Depicted is a bar graph showing the totalneurite length (μM) of cortical neurons treated with mock or 1 nM, 10nM, 100 nM, 1 μM, or 10 μM midi-GAGR in the presence of 25 μM 4-HNE(mean±SEM, n=40 cells. One way ANOVA, Bonferroni post hoc test, **:p<0.05; ****: p<0.0001).

FIG. 13B: Midi-GAGR has a neurite outgrowth enhancing effect in thepresence of 200 μM H₂O₂. Depicted is a bar graph showing the totalneurite length (μM) of cortical neurons treated with mock or 1 nM, 10nM, 100 nM, 1 μM, or 10 μM midi-GAGR in the presence of 200 μM H₂O₂(mean±SEM, n=3 cells. One way ANOVA, Bonferroni post hoc test, *:p<0.05; ***: p<0.0001).

FIGS. 14A-14F: Midi-GAGR enhances neuritogenesis and actin filopodiaformation in differentiated neuro2A (N2A) cells. N2A cells were starvedin serum-free medium plus mock (FIG. 14A) or 1 μM midi-GAGR (FIG. 14B)for 3 days and stained by antibody to α-tubulin (green) and phalloidin(actin: red). The actin filopodia were observed in neurites ofmidi-GAGR-treated cells (FIG. 14D), but not in mock treated cells (FIG.14C). After treatment with mock or midi-GAGR, cells were treated withmock (FIG. 14E) or 100 μM H₂O₂ (FIG. 14F) for one day and processed forimmunocytochemistry as described above. (Scale bars in FIG. 14A-FIG.14F=75 μm.)

FIG. 15A: Midi-GAGR treated N2A cells form neurites in the presence of25 μM 4-HNE and 200 μM H₂O₂. Depicted is a bar graph showing the numbersof neurites in N2A cells starved in serum-free-medium plus mock or 1 μMmidi-GAGR for 3 days (n=90, 30 cells×3 independent experiments,mean±SEM, *=p<0.001).

FIG. 15B: Midi-GAGR treated N2A cells form neurites in the presence of25 μM 4-HNE and 200 μM H₂O₂. Depicted is a bar graph showing the averagetotal neurite lengths of N2A cells starved in serum-free medium plusmock or 1 μM midi-GAGR for 3 days and then treated with mock, 100 or 200μM H₂O₂, or 25 or 50 μM 4-HNE (n=90, 30 cells×3 independent experiments,mean±SEM, * (blue star)=p<0.001 compared to mock, * (red star)=p<0.001compared to treatment with free radicals minus midi-GAGR.

FIG. 16: Midi-GAGR enhances total neurite length of primary corticalneurons. Depicted is a bar graph showing the average total neuritelength (μM) of primary cortical neurons treated with mock or midi-GAGR,low acyl gellan gum (LA), high acyl gellan gum (HA), dextran, oralginate (mean±SEM).

FIG. 17: In vitro blood-brain barrier (BBB) filter system. Depicted is aschematic showing the generation of an in vitro BBB consisting of bEND3cells and astrocytes.

FIG. 18: Midi-GAGR penetrates an in vitro blood-brain barrier (BBB).Depicted is a bar graph showing the average total neurite length of N2Acells starved for 3 days in serum-free medium without (no F) or underthe in vitro BBB filter system (F) on which mock or 1 or 10 μM midi-GAGRwas added and then treated with mock or 25 μM 4-HNE (n=60, 30 cells×2independent experiments, mean±SEM, *=p<0.001).

FIG. 19: Midi-GAGR binds to NCAM1. Depicted is a bar graph showing thenormalized spectrum count for mock (control) or midi-GAGR binding toneurexin, carboxypeptide D, or NCAM1. Data is the result of twoindependent experiments.

FIG. 20A: Mini- and midi-GAGR enhance osteogenesis of adult mesenchymalstem cells. Human bone marrow-derived mesenchymal stem cells werecultured in osteogenic media and α MEM media on 24-well plates for 14day. Cells were treated with control, 1 μM mini-GAGR or 1 μM midi-GAGRevery two days.

FIG. 20B: Mini- and midi-GAGR enhance osteogenesis of adult mesenchymalstem cells. Human bone marrow-derived mesenchymal stem cells werecultured in osteogenic media and α MEM media on 24-well plates for 14day. Cells were treated with control, 1 μM mini-GAGR or 1 μM midi-GAGRevery two days. After 14 days, cells were stained with Alizarin S andphotographed using a light phase microscope.

FIGS. 21A-21B: Mini- and midi-GAGR enhance osteogenesis of adultmesenchymal stem cells. Human bone marrow-derived mesenchymal stem cellswere cultured in osteogenic media and α MEM media on 24-well plates for14 day. Cells were treated with control, 0.1 μM mini- or midi-GAGR, or 1μM mini- or midi-GAGR (FIG. 21A). After 14 days, cells were stained withAlizarin S and photographed using a light phase microscope (FIG. 21B).

FIGS. 22A-22I: Midi-GAGR reverses neurite atrophy caused by 4HNE andH₂O₂. Differentiated N2A cells were treated with differentconcentrations of 4HNE (0, 1, 5, 10, and 25 μM) for 48 h or H₂O₂ (0, 1,10, 50, 100, and 200 μM) for 24 h and immunostained with α-tubulinantibody. The representative images of N2A cells treated with H₂O(vehicle) (FIG. 22A), 25 μM 4HNE (FIG. 22B), or 200 μM H₂O₂ (FIG. 22C).Scale bar=75 μm. Bar graphs represent the average total neurite lengthsof N2A cells in response to different concentrations of either 4HNE(FIG. 22D) or H₂O₂ (FIG. 22E). *, p<0.05 and **, p<0.001 compared tocontrol. (FIG. 22F-FIG. 22I.) Differentiated N2A cells were pre-treatedwith different concentrations (0, 0.001, 0.01, 0.1, 1, and 10 μM) ofmidi-GAGR for 24 h, followed by incubation with either 25 μM 4HNE for 48h or 200 μM H₂O₂ for 24 h and then immunostained with anti-α-tubulinantibody. The representative images of N2A cells pretreated with 1 μMmidi-GAGR and then incubated with either 25 μM 4HNE (FIG. 22F) or 200 μMH₂O₂ (FIG. 22G). Scale bar=75 μm. Bar graphs show the average totalneurite lengths of N2A cells pre-treated with different concentrationsof midi-GAGR followed by treatment with 25 μM 4HNE (FIG. 22H) or 200 μMH₂O₂ (FIG. 22I). Dotted lines correspond to the average total neuritelengths of N2A cells without any treatment. Data represent mean±SEM of,at least, 40 cells/group from two independent experiments. *, p<0.05 and**, p<0.001 compared to 4HNE alone.

FIGS. 23A-23G: Pre-treatment with midi-GAGR significantly reduces theapoptosis of rodent cortical neurons caused by 4HNE, H₂O₂, and amyloid βpeptide. Mouse cortical neurons at DIV5 were pre-treated with H₂O(vehicle), 1 μM midi-GAGR, 0.1 μM LA-GAGR, 0.01 μM HA-GAGR, 1 μMalginate, or 1 μM dextran followed by incubation with vehicle, 10 μM4HNE, or 50 μM H₂O₂ for 24 h or 2 μM Aβ₄₂ for 48 h. After the treatment,neurons were processed for live/dead assay using calcein AM and ethidiumhomodimer-I. The representative images of H₂O (FIG. 23A), 4HNE alone(FIG. 23B), 4HNE+midi-GAGR (FIG. 23C), H₂O₂ alone (FIG. 23D),H₂O₂+midi-GAGR (FIG. 23E), Aβ₄₂ alone (FIG. 23F), and Aβ₄₂+midi-GAGR(FIG. 23G). Live cells were labeled as green and dead cells as red.Scale bar=100 μm. Insets show the magnified images of individualneurons. Inset scale bar=50 μm.

FIGS. 24A-24C: Quantification of neuron death in response to oxidativeinsults in the presence of different polysaccharides. The numbers oflive and dead cells were counted using Metamorph software. Bar graphsrepresent the percents of dead neurons after the pre-treatments withdifferent polysaccharides followed by treatment with 4HNE (FIG. 24A),H₂O₂ (FIG. 24B), or Aβ₄₂ (FIG. 24C). Data represent mean±SEM of threeindependent experiments. For each experiment, at least, 1,000 cells pergroup were counted. *, p<0.05.

FIGS. 25A-25B: Midi-GAGR protects rodent cortical neurons fromco-treated 4HNE, Aβ₄₂ peptide, and activated microglial cells. (FIG.25A.) Rat cortical neurons (E17) at DIV5 were co-treated with either 10μM 4HNE (for 24 h) or 2 μM Aβ₄₂ (for 48 h) and either water or 1 μMmidi-GAGR. After the treatment, neurons were processed for live/deadassay using calcein AM and ethidium homodimer-I. Live and dead cellswere imaged using a fluorescence microscope. The numbers of live anddead cells were counted using Metamorph software. Bar graphs show thepercents of dead neurons after co-treatment with either 4HNE or Aβ₄₂plus/minus midi-GAGR. Data represent mean±SEM of three independentexperiments. *, p<0.05. (FIG. 25B.) The co-cultures of rat corticalneurons and microglia cells were treated with 2 μM Aβ₄₂ plus/minus 1 μMmidi-GAGR. After 48 h, transwell filters containing microglial cellswere removed and neurons in bottom wells were processed for live/deadassay. Live and dead cells were imaged using a fluorescence microscope.The numbers of live and dead neurons were counted using Metamorphsoftware. Bar graphs show the percent of dead neurons. Data representmean±SEM of three independent experiments. *, p<0.05.

FIGS. 26A-26C: Midi-GAGR enhances neurite outgrowth in N2A cells.Differentiated N2A cells were treated with different concentrations ofmidi-GAGR for 48 h and immunostained using anti-α-tubulin antibody.Representative confocal images of N2A cells treated with either H₂O(vehicle) (FIG. 26A) or 1 μM midi-GAGR (FIG. 26B). Scale bar=30 μm. Bargraphs in FIG. 236C show the average total neurite lengths of N2A cellstreated with different concentrations of midi-GAGR (mean±SEM of, atleast, three independent experiments). *, p<0.05 compared to control.

FIGS. 27A-27G: Midi-GAGR enhances neuritogenesis in mouse corticalneurons. Mouse cortical neurons (E17, DIV4) were treated with H₂O(vehicle) (FIG. 27A), 1 μM midi-GAGR (FIG. 27B), 0.1 μM LA-GAGR (FIG.27C), 0.01 μM HA-GAGR (FIG. 27D), 1 μM alginate (FIG. 27E), or 1 μMdextran (FIG. 27F) for 48 h and immunostained with anti-α-tubulinantibody. Scale bar=100 μm. (FIG. 27G.) Bar graphs show average foldchanges in the total neurite length of mouse cortical neurons inresponse to different polysaccharides (mean±SEM of, at least, threeindependent experiments). **, p<0.01 compared to control.

FIGS. 28A-28F: Midi-GAGR activates CREB, a neurotrophic transcriptionalfactor. Mouse cortical neurons (DIV4) were treated with H₂O (vehicle), 1μM midi-GAGR, 0.1 μM LA-GAGR, 0.01 μM HA GAGR, 1 μM alginate, or 1 μMdextran for 48 h and immunostained with DAPI (not shown) and theantibodies to α-tubulin and pCREB. The representative images of neuronstreated with H₂O (FIG. 28A), midi-GAGR (FIG. 28B), alginate (FIG. 28C)or dextran (FIG. 28D), followed by staining with α-tubulin (green) andpCREB (red). Scale bar=30 μm. Bar graphs show the average intensities ofpCREB after different treatments (n=90 neurons, mean±SEM of, at least,three independent experiments). *, p<0.05 compared to control. (FIG.28E.) The cytosols extracted from neurons treated with H₂O (vehicle), 1μM midi-GAGR, 0.1 μM LA-GAGR, 0.01 μM HA GAGR, 1 μM alginate, or 1 μMdextran for 48 h were used for immunoblotting using the antibodies topCREB and CREB (neurons extracted from sixteen E17 mouse embryos, n=2experiments). (FIG. 28F.)

FIGS. 29A-29C: Intranasally administered midi-GAGR increases theexpression of NF200 and GAP-43 in the brains of live rats. SD rats wereintranasally administered with either vehicle or midi-GAGR and processedto obtain brains at 6 h, 24 h, or 48 h after the administration. Brainswere dissected to the frontal cortex, hippocampus, and rest of thebrain. (FIG. 29A.) Brain tissue lysates were processed forimmunoblotting using the antibody to NF200 (upper panel), GAP-43 (middlepanel), or GAPDH (lower panel). ‘C’ is control and ‘M’ is midi-GAGR. Theband densities of NF200 and GAP-43 were measured using image J softwareand normalized to those of GAPDH. Bar graphs show fold changes in thelevel of NF200 (FIG. 29B) and GAP-43 (FIG. 29C) in the different partsof brains at given time points. Data represents mean±SEM (n=4animals/group). *, p<0.05.

FIGS. 30A-30K: Midi-GAGR binds to FGFR1 and uses FGFR1 signaling pathwayto activate CREB and protect neurons from the death caused by oxidativeinsult. FIG. 30A: Midi-GAGR- or dextran-conjugated epoxy sepharose beadswere mixed with synaptosomal plasma membrane proteins in 0.5% IgepalCA-630 PMEE buffer to pull down midi-GAGR-interacting FGFR1.Precipitated FGFR1 was detected by immunoblotting (n=2, four ratbrains). FIGS. 30B-J: Mouse cortical neurons (DIV4) were pre-treatedwith H₂O (vehicle, FIGS. 30B-30C) or the inhibitors of FGFR1 (SU5402[SU], 4 μM, FIG. 30D, PKC (staurosporine [Stau], 3 nM, FIG. 30E), MEK(U0126 [U01], 10 μM, FIG. 30F), PI3K (LY294002 [LY], 20 μM, FIG. 30G),CaMKII (KN-62 [KN], 10 μM, FIG. 30H), or FAK (PF-573228 [PF], 1 μM, FIG.30I) for 6 h and then with mock (FIG. 30B) or 1 μM midi-GAGR (+midi,FIGS. 30C-30I) for 48 h. Neurons were then immunostained with theantibodies to α-tubulin (red) and p-CREB (green). Scale bar=100 μm.(FIG. 30J.) Bar graphs show the average intensities of pCREB afterdifferent treatments (n=60 neurons, mean±SEM). *, p<0.01 and #, p<0.05compared to control. (FIG. 30K.) Rat cortical neurons (E17, DIV6) weretreated for 6 h with FGFR1 inhibitor (SU5402, 4 μM) and treated with 10μM 4HNE and either vehicle or 1 μM midi-GAGR for 24 h prior to cellviability/cytotoxicity assay. As controls, neurons were treated with4HNE, 4-HNE plus midi-GAGR, SU5402, midi-GAGR, or SU5402 plus midi-GAGR.Live and dead cells were imaged using a fluorescence microscope. Thenumbers of live and dead neurons were counted using Metamorph software.Bar graphs show the percent of dead neurons. Data represent mean±SEM ofthree independent experiments. For each experiment, at least, 200 cellsper group were counted. *, p<0.05 (n.s.: not significant)

FIGS. 31A-31I: Intranasally administered midi-GAGR increased neuronalactivity markers and decreased hyperphosphorylated tau in 3×Tg-AD mice.FIG. 31A: 12-week-old 3×Tg-AD mice were intranasally administered with40 μL (20 μL/nostril) of either sterile H₂O (Veh.) or 7.4 mM midi-GAGR(midi) every day for 14 days and processed to obtain the cortex andhippocampus (Hippo). The tissues were homogenized to extract proteinsfor immunoblotting using the antibodies to NF200, GAP-43, PSD95,synaptophysin, pCREB, CREB, p-tau (AT8), tau, and GAPDH. (FIGS. 31B-31I)The densities of protein bands were measured using Image J andnormalized to that of the loading control, GAPDH. Normalized values wereused to calculate the average normalized band densities of NF200 (FIG.31B), GAP-43 (FIG. 31C), PSD95 (FIG. 31D), synaptophysin (SYN, FIG.31E), pCREB (FIG. 31F), total CREB (FIG. 31G), p-tau (FIG. 31H), andtotal tau (FIG. 31I). Data represent mean±SEM of three independentanimals. #, p<0.01, *, p<0.05.

FIGS. 32A-32B: 3×Tg-AD mice were treated either with sterile water(vehicle) or 0.296 nmole of midi-GAGR for 14 days. White feathery paperswere scattered at 0 hr. After 24-h post treatment, the image of thecages was taken by a camera (FIG. 32A). The daily sizes (areas) of thenests made by the mice were measured using software and used tocalculate average nest size (FIG. 32B).

FIG. 33: Human umbilical vein endothelial cells (HUVECs) were incubatedon EGM with 0, 0.1, or 1 μM of mini-GAGR or midi-GAGR for 16-18 h,fixed, and labeled with Acridine Orange. The tubes formed from HUVECswere imaged by fluorescent microscopy. The total length of tubes formedfrom HUVECs per image was measured by ‘Metamorph’ software (n=4experiments).

FIGS. 34A-34B: Human bone marrow-derived mesenchymal stem cells(BM-MSCs) in cartilage-forming medium were treated with none ormini-/midi-GAGR (0.1-1 μM) every 2 days in a 24 well plate. On day 14,GAGs were stained with 1% Alcian Blue. The images of Alcian Bluestaining of the whole well or individual spheroids were taken by a lightmicroscope. (FIG. 34A.) On day 7, cytosols were obtained in 1% NP-40PMEE buffer from cartilage cells and used for immunoblotting usingantibodies to type II collagen and beta-actin (n=2). (FIG. 34B.)

FIG. 35: Dextran or mini-GAGR was conjugated to epoxy sepharose beadsaccording to manufacturer's protocol. The beads were then incubated inthe plasma membrane extractions of N2A cells after those were blocked in2% BSA. FGFR1 protein that bound to the beads was detected byimmunoblotting using anti-FGFR1 antibody (n=2).

FIGS. 36A-36B: Human BM-MSCs were treated with 1 (control: H₂O), 0.01,0.1, or 1 μM of midi-GAGR or mini-GAGR for 14 days in adipogenic medium.Thereafter, differentiated or undifferentiated cells were stained withOil-red O and imaged using light microscopy.

FIG. 37A: The standard curve of emissions at 520 nm (relativefluorescence unit: RFU) of ANTS at 0, 0.1, 0.3, 1, 3, and 10 mM (n=5).

FIG. 37B: The standard curve of absorbances at 490 nm for 0, 7.4, and 74nM, 0.74 and 7.4 mM of midi-GAGR (n=3).

FIG. 37C: Fluorescent and colorimetric values of ANTS-midi-GAGR beforeand after three 75% ethanol washes (mean±standard error, n=5).

FIG. 38A: The RFUs (mean±standard error) of the brain cytosols fromanimals administered with none or 1 mM ANTS-midi-GAGR. Brains weredissected out of rats that were sacrificed at 6 h after administration(n=3).

FIG. 38B: The RFUs (mean±standard error) of the sera from animalsadministered with none or 1 mM ANTS-midi-GAGR. Serum samples werecollected from rats that were sacrificed at 6 h after administration(n=4).

FIG. 38C: The RFUs (mean±standard error) of the supernatants and pelletsof TCA precipitation and ethanol precipitation of sera (n=5).

FIG. 39: Schematic illustration showing a method to examine theBBB-permeability and tissue distribution of a target polysaccharide inanimal. A polysaccharide was conjugated with fluorescent8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS) fortracking in animals. ANTS-tagged polysaccharide was separated fromunconjugated free ANTS using 75% ethanol. After ANTS-polysaccharide wasintra-nasally administered into animals, the amounts ofANTS-polysaccharide in the brain and the serum were quantified byfluorocytometry. Free ANTS-polysaccharide was separated from serumproteins using 75% ethanol and trichloroacetic acid (TCA).

DETAILED DESCRIPTION OF THE INVENTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced. The disclosures of thesepublications, patents and published patent specifications are herebyincorporated by reference into the present disclosure to more fullydescribe the state of the art to which this invention pertains.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. The term“comprises” means “includes.” The abbreviation, “e.g.” is derived fromthe Latin exempli gratia, and is used herein to indicate a non-limitingexample. Thus, the abbreviation “e.g.” is synonymous with the term “forexample.”

Definitions

As used herein, the term “LA-GAGR” refers to low acyl gellan gum.LA-GAGR is a polysaccharide based on a tetrasaccharide repeating unitconsisting of glucose derivatives such as glucuronic acid, mannose, andrhamnose connected by (1-3)-α and (1-4)-β, and has an average molecularweight of 99,600 g/mol (LA-GAGR:[D-Glc(β1→4)D-GlcA(β1→4)D-Glc(β1→4)L-Rha(α1→3]n) (FIG. 1). The molecularweights of the repeating units are reported to be 700-1,000 g/mol,depending on their functional groups and branches. Gellan gum isproduced by fermentation of calcium hydrates in Sphingomonas elodeamonocultures extracted from natural resources. Primarily used as a foodadditive, LA-GAGR may be found in food products such as baked goods,cake icings, various sweets, jellies and spreads, jams, puddings,sauces, dairy products, and microwave-ready foods. LA-GAGR can also beused in cosmetic and hygiene products such as makeup, facial masks,creams, and lotions. In pharmaceuticals, LA-GAGR is used to make tabletsthat are easy to swallow, as well as to adjust the rate of release ofmedicinal compounds in the body.

As used herein, “GAGR” generally refers to gellan gum which has amolecular weight greater than that of native gellan gum. The term“native gellan gum” generally refers to a gellan gum that has not beenmodified by physical or chemical means.

As used herein, the term “neurodegenerative disorders” refers to anycentral nervous system (CNS) or peripheral nervous system (PNS) diseasethat is associated with neuronal or glial cell defects including but notlimited to neuronal loss, neuronal degeneration, neuronal demyelination,gliosis (i.e., astrogliosis), or neuronal or extra-neuronal accumulationof aberrant proteins or toxins (e.g., β-amyloid, or α-synuciein). Theneurological disorder can be chronic or acute. Non-limiting examples ofvarious chronic and acute neurological diseases include Parkinson'sdisease, Alzheimer's disease, amyotrophic lateral sclerosis, myastheniagravis, multiple sclerosis, microbial infections, stroke, Pick'sdisease, dementia with Lewy bodies, Huntington disease, chromosome 13dementias, Down's syndrome, cerebrovascular disease, Rasmussen'sencephalitis, viral meningitis, NPSLE, amyotrophic lateral sclerosis,Creutzfeldt-Jacob disease, Gerstrnann-Straussler-Scheinker disease,transmissible spongiform encephalopathies, ischemic reperfusion damage(e.g., stroke), brain trauma, spinal cord injury, microbial infection,chronic fatigue syndrome, Mild Cognitive Impairment, movement disorders(including ataxia, cerebral palsy, choreoathetosis, dystonia, Tourette'ssyndrome, kernicterus), tremor disorders, leukodystrophies (includingadrenoleukodystrophy, metachromatic leukodystrophy, Canavan disease,Alexander disease, Pelizaeus-Merzbacher disease); neuronal ceroidlipofucsinoses, ataxia telangectasia, and Rett Syndrome.

As used herein, the term “neuroprotective” refers to the relativepreservation of neuronal structure and/or function. In a case of ongoingneurodegenerative insult, the relative preservation of neuronalintegrity implies a reduction in the rate of neuronal loss over time.Neuroprotection aims to prevent or slow disease or injury progressionand secondary injuries by halting or at least slowing the loss ofneurons.

As used herein, the term “bone disorders” refers to a conditioncharacterized by altered bone metabolism. Non-limiting examples includeosteoporosis, including post-menopausal osteoporosis, osteopenia,Paget's disease, osteolytic metastasis in cancer patients,osteodistrophy in liver disease and the altered bone metabolism causedby renal failure or haemodialysis, osteogenesis imperfecta, bonefracture, bone surgery, aging, pregnancy, and malnutrition.“Osteogenesis” refers to the formation and development of bone.

As used herein, the term “effective amount” refers to an amount of anagent that is sufficient to effect a therapeutically significantneuroprotective effect, a therapeutically significant neurotrophiceffect, or alternatively, a therapeutically significant osteogeniceffect, in a subject diagnosed with a neurodegenerative disorder or bonedisorder, respectively. The therapeutically effective amounts to beadministered will depend on the severity of the condition and individualsubject parameters including age, physical condition, size, weight, andconcurrent treatment. In certain embodiments, a maximum dose can beused, that is, the highest safe dose according to sound medicaljudgment. However, a lower dose or tolerable dose may be administeredfor medical reasons, psychological reasons, or for virtually any otherreason.

The actual dosage amount of a composition administered to the subject,such as a human subject, can be determined by physical and physiologicalfactors such as body weight, severity of condition, the type of diseasebeing treated, previous or concurrent therapeutic interventions,idiopathy of the subject and on the route of administration. Thepractitioner responsible for administration will, in any event,determine the concentration of active ingredient(s) in a composition andappropriate dose(s) for the individual subject.

As used herein, the term “therapeutic agent” refers to any biologicagent, molecule, compound, and/or substance that is used for the purposeof treating and/or managing a disease or disorder. As used herein,“agent” refers to any biologic agent, molecule, compound, methodology,and/or substance for use in the prevention, treatment, management,and/or diagnosis of a disease or disorder. As used herein, the terms“prevent,” “preventing,” and “prevention” in the context of theadministration of a therapy to a subject refer to the prevention orinhibition of the recurrence, onset, and/or development of a disease orcondition, or a symptom thereof, in a subject resulting from theadministration of a therapy (e.g., a prophylactic or therapeutic agent),or a combination of therapies (e.g., a combination of prophylactic ortherapeutic agents).

As used herein, the terms “treat,” “treatment,” and “treating” in thecontext of the administration of a therapeutic agent to a subject referto the reduction or inhibition of the progression and/or duration of adisease or condition, the reduction or amelioration of the severity of adisease or condition, and/or the amelioration of one or more symptomsthereof resulting from the administration of one or more therapies.

The terms “subject,” “individual,” and “patient” are defined herein toinclude animals such as mammals, including but not limited to, primates,cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, miceor other bovine, ovine, equine, canine, feline, rodent, or murinespecies. In one embodiment, the subject is a mammal (e.g., a human)afflicted with a neurodegenerative disorder. In another embodiment, thesubject is a mammal (e.g., a human) afflicted with a bone disorder.

The term “gel” refers to a jelly-like material composed of across-linked system.

Pharmaceutical compositions are characterized as being sterile andpyrogen-free. As used herein, “pharmaceutical compositions” includecompositions for human and veterinary use. Methods for preparingpharmaceutical compositions of the invention are within the skill in theart, for example, as described in Remington's Pharmaceutical Science,17th ed., Mack Publishing Company, Easton, Pa. (1985), the entiredisclosure of which is incorporated herein by reference.

Pharmaceutical compositions can also comprise conventionalpharmaceutical excipients and/or additives. Suitable pharmaceuticalexcipients include stabilizers, antioxidants, osmolality adjustingagents, buffers, and pH adjusting agents. Suitable additives includephysiologically biocompatible buffers (e.g., tromethaminehydrochloride), chelants (such as, e.g., DTPA or DTPA-bisamide) orcalcium chelate complexes (e.g., calcium DTPA, CaNaDTPA-bisamide), or,optionally, additions of calcium or sodium salts (e.g., calciumchloride, calcium ascorbate, calcium gluconate, or calcium lactate).Pharmaceutically-acceptable carriers can include water, buffered water,normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid, and the like.Pharmaceutical compositions can be packaged for use in liquid form, orcan be lyophilized.

For solid pharmaceutical compositions, nontoxic solidpharmaceutically-acceptable carriers can be used; for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, and the like. For example, a solid pharmaceutical compositionfor oral administration can comprise any of the carriers and excipientslisted above and 10-95%, preferably 25%-75%, of the neuroprotective orosteogenic agent. A pharmaceutical composition for aerosol(inhalational) administration can comprise 0.01-20% by weight,preferably 1%-10% by weight, of neuroprotective or osteogenic agentencapsulated in a liposome, as described above, and a propellant. Acarrier can also be included as desired, e.g., lecithin, for intranasaldelivery.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the active compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from at least about 1microgram/kg/body weight, including about 5, about 10, about 50, about100, about 200, about 350 weight, about 500 microgram/kg/body weight; orat least about 1 milligram (mg)/kg/body weight, such as about 5, about10, about 50, about 100, about 200, about 350, about 500, to about 1000mg/kg/body weight or more per administration, and any range derivabletherein.

General Description

Described herein are methods for generating midi- and mini-GAGR,compositions comprising midi- and mini-GAGR, and applications andmethods employing such compositions.

In particular embodiments described herein are methods of generatingdigestion products of low acyl gellan gum (LA-GAGR). In theseembodiments, LA-GAGR is enzymatically digested by α1→3 glucosidase.Allowing the digestion to proceed for 24, 48, 72 hours, or longerproduces three different digestion products: maxi-GAGR, midi-GAGR, andmini-GAGR, respectively.

Low acyl gellan gum (LA-GAGR) is a polysaccharide consisting of arepeating tetrasaccharide,D-Glc(β1→4)D-GlcA(β1→4)D-Glc(β1→4)L-Rha(α1→3). FIG. 1A shows thechemical structure of LA-GAGR([D-Glc(β1→4)D-GlcA(β1→4)D-Glc(β1→4)L-Rha(α1→3]n). Arrows point toeither α- or β-glucosidic bonds. Low acyl gellan gum is used as foodadditive, capsule for drug delivery, and coating for surgical device inhuman. Low acyl gellan gum shows few side effects in human.

LA-GAGR can be enzymatically digested by α(1→3) glucosidase to generateseveral digestion products. A 24 hour enzymatic digestion of LA-GAGRresults in a digestion product having an average molecular weight ofapproximately 30,245 g/mol. “midi-GAGR” is generated after 48 hoursenzymatic digestion of LA-GAGR. Midi-GAGR has a molecular weight ofapproximately 4,774 g/mol “mini-GAGR” is generated after ≥72 hoursenzymatic digestion of LA-GAGR. Mini-GAGR has a molecular weight ofapproximately 718 g/mol, which is close to the molecular weight of thebasic repeating sugar units of LA-GAGR. Both mini-GAGR and midi-GAGR arebelieved to have few side effects in human. LA-GAGR may be similarlydigested by other glucosidases, for example, α(1→4) glucosidase.

In other embodiments described herein are compositions comprisingmidi-GAGR and/or mini-GAGR. In accordance with the present disclosure,the compositions described herein have been found to be neuroprotective.In certain specific embodiments, the compositions described herein canbe used according to methods described herein to treat neurodegenerativedisorders, e.g., Alzheimer's disease, Parkinson's disease, multiplesclerosis, amyotrophic lateral sclerosis, spinal cord injury, and braintrauma. In other embodiments, the compositions are useful for woundhealing, promoting collagen production, and promoting blood vesselformation.

In yet other embodiments described herein are methods for treatingdegenerative neurological or disorder, healing wounds, stimulatingcollagen production, activating FGF receptors, or stimulating bloodvessel formation using the compositions described herein.

While not wishing to be bound by any particular theory, theneuroprotective effect of midi-GAGR is now believed to be attributableto its antioxidant activity, and its ability to bind and interact withfibroblast growth factor receptor 1 (FGFR1), neural cell adhesionmolecule 180 (NCAM-180), and neurofascin-186. Because of midi-GAGR'santioxidant activity and ability to bind and interact with NCAM-180,compositions described herein comprising midi-GAGR can be used accordingto the methods described herein for the treatment of degenerativeneurological disorders.

In other particular embodiments described herein, compositionscomprising midi- and/or mini-GAGR are useful to enhance osteogenesis ofadult mesenchymal stem cells. In certain specific embodiments, thecompositions described herein can be used according to methods describedherein to treat bone disorders, e.g., oseteoporosis, osteoarthritis,osteogenesis imperfect, and bone fractures. In one embodiment, theosteogenic effect of midi- and mini-GAGR is attributable to thepolysaccharides enhancing bone cell formation from human adultmesenchymal cells under osteogenic conditions at low concentrations.Because of midi- and mini-GAGR's osteogenic effect, compositionsdescribed herein comprising midi- and/or mini-GAGR can be used accordingto methods described herein for the treatment of bone disorders.

Wound healing is a complex and dynamic process of replacing devitalizedand missing cellular structures and tissue layers. It is mediated by acomplex and coordinated series of events such as inflammatory,fibroblastic, and remodeling phases. In addition to the phases,angiogenesis (neo-vascularization), re-epithelization, andproduction/deposition of new glycosaminoglycans (GAGs) and proteoglycansat a wounded site are important for complete wound healing. Variousprotein growth factors are used in healing dressing to enhance woundhealing. Among the protein growth factors, fibroblast growth factors(FGFs) show noteworthy ability in tissue repair and regeneration, evenas a component of wound healing dressing. In spite of this ability, theclinical use of FGFs is limited by their low thermal stability and highsensitivity to proteases. Similarly to FGFs, other protein growthfactors are also limited by the same problems. Thus, an alternativewound-healing agent has been searched for.

In accordance with the present disclosure, the strong vasculogenicfactors mini-GAGR (the basic unit) and midi-GAGR, which are the cleavageproducts of low acyl gellan gum (FDA 21 CFR 172.665), are useful forwound healing. These cleavage products have strong enhancing effects onblood vessel formation (i.e., strong angiogenic effect) and tissuematrix production, and interact with the FGF receptor. Midi- andmini-GAGR increase the production of GAG and collagen, the major tissuecomponents that refill wound sites. In addition, these GAGR cleavageproducts do not cause fat formation. Fat hypertrophy interferes withwound healing. All of this demonstrates that these cleavage products areuseful as therapeutic agents for wound healing.

In the examples herein, treatment with 0.1-1 μM mini-GAGR and midi-GAGRdrastically enhanced blood vessel formation from human umbilical veinendothelial cells (HUVECs) (FIG. 33). Moreover, both mini-GAGR andmidi-GAGR increased the production of GAG and collagen, the major tissuecomponents that refill wound sites (FIG. 34). Without wishing to bebound by theory, it is believed that these vasculogenic andGAG-producing effects of mini-GAGR and midi-GAGR are exerted by theirinteraction with FGF receptor 1 (FGFR1) that binds FGFs and has beenshown to be important for wound healing (FIG. 35). In addition,mini-GAGR and midi-GAGR did not enhance cell proliferation andadipogenesis (FIG. 36), indicating that both do not cause tumorigenesisand tissue adipogenesis. Thus, midi- and mini-GAGR have neithermitogenic effect nor adipogenic effect. Importantly, midi-GAGR (likelymini-GAGR, too) shows a long functional half-life (˜24 hours) in thebody, indicating that they are thermo-stable and degradation-resistantinside the body; that is, they overcome the limitation of proteinfactors. Moreover, the examples herein show that midi- and mini-GAGR canform blood vessels and extracellular matrix to an extent similar toprotein growth factors. Taken together, this demonstrates thatmidi-GAGR, mini-GAGR, and even their precursor, low acyl gellan gum,have a significant therapeutic ability for wound healing treatment.Moreover, since low acyl gellan gum is already used in human(FDA-approved), it can be readily used for the clinical application inhumans.

LA-GAGR and/or its cleavage products midi-GAGR and mini-GAGR can beformulated in a gel product for wound healing applications. In someembodiments, the gel formula can controllably release an effective doseof the LA-GAGR, midi-GAGR, or mini-GAGR at a skin wound site on asubject, such as a human or animal. LA-GAGR and/or its cleavage productsare easily encapsulated in a clinical gel for application onto woundsites, and provide the additional benefit of an anti-fouling property.To formulate a gel, the polysaccharide component of LA-GAGR and/or itscleavage products is/are bound to a gelling component. Non-limitingexamples of suitable gelling components are chitosan, alginate,hyaluronic acid, hyaluronates, collagen, xanthan gum, pectins,carboxymethylcellulose, arabic gum, and/or the like.

GAGR and/or its cleavage products can be incorporated into the varioustypes of wound dressings currently available, such as hydrogeldressings, perforated plastic film absorbent dressings, hydrocolloiddressings, or alginate dressings. Most commercially available geldressing do not contain a wound healing agent, but rather merely providea scaffold for tissue regeneration. Wound healing agents are typicallyexpensive and require a special encapsulation system for focal deliveryat the wound site. However, LA-GAGR and its cleavage products arecost-effective and are easily incorporated into a gel dressing. Thus,LA-GAGR and/or its cleavage products can provide for an efficient geldressing that includes a wound healing agent.

Gel compositions containing LA-GAGR or its cleavage product naturallyrelease the LA-GAGR or its cleavage product over time. Once applied, thegel degrades naturally over time, causing the release of the LA-GAGR orcleavage product.

In certain embodiments of a gel formulation, the formulation includesmini-GAGR. Without wishing to be bound by theory, it is believed thatbecause mini-GAGR is smaller than midi-GAGR, mini-GAGR diffuses better.In certain embodiments of a gel formulation, the formulation includesmidi-GAGR. Without wishing to be bound by theory, it is believed thatmidi-GAGR has a higher antioxidant capacity than mini-GAGR, and istherefore more efficient than mini-GAGR.

Therapeutic/Prophylactic Methods and Compositions

Further described herein are methods of treatment and prophylaxis byadministration to a subject an effective amount of a therapeuticcompound, i.e., mini- and/or midi-GAGR. In a preferred aspect, thetherapeutic is substantially purified. The subject is preferably ananimal, including but not limited to, animals such as cows, pigs,chickens, etc., and is preferably a mammal, and most preferably human.

Various delivery systems are useful to administer a therapeuticcompound, e.g., encapsulation in liposomes, microparticles,microcapsules, etc. Methods of introduction include, but are not limitedto, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, and oral routes. The therapeutic compounds areadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer thetherapeutic compositions locally to the area in need of treatment; thismay be achieved by, for example, and not by way of limitation, localinfusion during surgery, topical application, e.g., in conjunction witha wound dressing after surgery, by injection, by means of a catheter, bymeans of a suppository, or by means of an implant, the implant being ofa porous, non-porous, or gelatinous material, including membranes, suchas sialastic membranes, or fibers.

Pharmaceutical Compositions

Such compositions comprise a therapeutically effective amount of atherapeutic, such as mini- and/or midi-GAGR, and a pharmaceuticallyacceptable carrier or excipient. Such a carrier includes, but is notlimited to, saline, buffered saline, dextrose, water, glycerol, ethanol,and combinations thereof. The carrier and composition can be sterile.The formulation will suit the mode of administration.

In one embodiment, pharmaceutical compositions disclosed herein comprisea neuroprotective agent, wherein the agent is LA-GAGR or its digestionproduct, mixed with a pharmaceutically acceptable carrier. In oneparticular embodiment, the agent is the digestion product midi-GAGR,mini-GAGR, or a combination thereof.

In another embodiment, the present pharmaceutical compositions comprisean osteogenic agent, wherein the agent is LA-GAGR or its digestionproduct, mixed with a pharmaceutically acceptable carrier. In oneparticular embodiment, the agent is the digestion product midi-GAGR,mini-GAGR, or a combination thereof.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. The composition can be aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc.

In one embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. For example, compositionsfor intravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition also includes a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it is be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline is provided so that the ingredients are mixed prior toadministration.

The therapeutic formulation can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with free aminogroups such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with free carboxyl groupssuch as those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the therapeutic formulation which will be effective in thetreatment of a particular disorder or condition will depend on thenature of the disorder or condition, and is determined by standardclinical techniques. In addition, in vitro assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the disease or disorder, and isdecided according to the judgment of the practitioner and each patient'scircumstances. However, suitable dosage ranges for intravenousadministration are generally about 20-500 micrograms of active compoundper kilogram body weight. Suitable dosage ranges for intranasaladministration are generally about 0.01 pg/kg body weight to 1 mg/kgbody weight. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

Kits

Also provided are pharmaceutical packs or kits comprising one or morecontainers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) is a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

Any of the compositions described herein may be comprised in a kit. Inone embodiment, provided herein is a pharmaceutical pack or kitcomprising one or more dosage units of LA-GAGR or its digestion productsufficient for one or more courses of treatment for an adult mesenchymalstem cell or wound healing. Associated with such pharmaceutical pack orkit are instructions for administering the LA-GAGR digestion product.Optionally associated with such pharmaceutical pack or kit is a noticein the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human consumption.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there is more than one component in the kit, the kitalso will generally contain a second, third or other additionalcontainer into which the additional components may be separately placed.However, various combinations of components may be comprised in a vial.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being one preferred solution.

However, the components of the kit may be provided as dried powder(s).When components are provided as a dry powder, the powder can bereconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container means. It mayalso include components that preserve or maintain the polysaccharidesmini- and midi-GAGR, or that protect against their degradation.

The compounds, methods, and kits of the current teachings have beendescribed broadly and generically herein. Each of the narrower speciesand sub-generic groupings falling within the generic disclosure alsoform part of the current teachings. This includes the genericdescription of the current teachings with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

EXAMPLES

Certain embodiments of the present invention are defined in the Examplesherein. It should be understood that these Examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions.

Example 1 Generation of Midi-GAGR and Mini-GAGR

Low acyl gellan gum (LA-GAGR) has an average molecular weight of 99,600g/mol, based on a tetrasaccharide repeating unit consisting of glucoseand glucose derivatives such as glucuronic acid, mannose, and rhamnoseconnected by (1-3)-α and (1-4)-β (LA-GAGR:[D-Glc(β1→4)D-GlcA(β1→4)D-Glc(β1→4)L-Rha(α1→3)]_(n)). Depending on theirfunctional groups and branches, the molecular weights of the repeatingunits are 700-1,000 g/mol.

LA-GAGR was enzymatically digested by enzymatic hydrolysis using α1→3glucosidase, generating midi-GAGR and mini-GAGR. The digestion wasperformed in 0.1M acetate buffer (pH 5) containing 1% salicin (cofactor)in glass tubes. A 1% salicin solution was prepared by dissolving 1 gsalicin in 100 ml of 0.1 M acetate buffer at pH 5. The salicin solutionwas then incubated for 6-8 minutes before use. The mixture was shakenlightly in an incubator at 37° C. and 80 rpm. At 24, 48, and 72 hoursafter the start of enzymatic hydrolysis, the glass tubes were removedfrom the incubator, placed in a hot water bath for 5 minutes, and thenmoved to an ice bath to stop the digestion reaction. The 24, 48, and 72hour digestion products were named mega-GAGR, midi-GAGR, and mini-GAGR,respectively. The samples were refrigerated until molecular weightmeasurement.

Prior to molecular weight measurement, samples were placed in a vacuumdryer at −60 cmHg gauge at 70° C. The tubes were removed from the vacuumdryer after approximately 8 ml of 0.1 M acetate buffer had evaporated. 1g of LA-GAGR digestion sample was re-suspended in 30 ml of distilledwater. Molecular weights of the resulting LA-GAGR fragments were thenmeasured using a Parallel Plate Rheometer (PPR, Rheometrics, Inc.)equipped with rheometer software (TA Orchestrator, TA Instrument, Inc.).Molecular weights of the LA-GAGR digestion products were determined onthe basis of the viscosity-storage modulus profiles of the samples usingthe RheoAnalyzer program developed by TomCoat Oy, Inc., Finland. Thevalidity of the RheoAnalyzer program was verified by running polystyrenestandard (NBS 706) on the PPR and determining the molecular weight ofNBS 706 from its viscosity profile. The results of the molecular weightmeasurements are show in Table 1.

TABLE 1 The molecular weights of LA-GAGR enzymatic digestions. GAGR48-hour 72-hour Before 24-hour Enzymatic Enzymatic Enzymatic EnzymaticDigestion: Digestion: Digestion Digestion midi-GAGR mini-GAGR Molecular99,639 30,245 4,774 718 Weight (g/mol)

The hydrolysis of LA-GAGR may be also carried out by chemical, physical,or other enzymatic means or any combination thereof.

The chemical hydrolysis of LA-GAGR may be carried out by treatment withan acid but is preferably carried out by treatment with an alkali. Thephysical hydrolysis may be carried out by shear.

The physical hydrolysis of LA-GAGR may be performed at high pressure(e.g., greater than 500 psi) through a small orifice. This processcauses the polymer to break into smaller segments. The homogenizationprocess may be repeated to achieve further reduction in the molecularweight of the polymer.

Sonication may be used to cleave water soluble LA-GAGR into midi-GAGRand mini-GAGR. This method involves exposing the polymer sample to highfrequency waves.

The use of gamma radiation from either cobalt or electron beam sourcesalso can cleave water soluble LA-GAGR into midi-GAGR and mini-GAGR. Themolecular weight reduction occurs most readily when the polymer is inthe hydrated, rather than dry, form. For liquid samples, radiationlevels from 0.25 to 5 Mrad provide significant reductions in molecularweight.

The hydrolysis of LA-GAGR may be performed by exposing the polymer to anoxidizing agent such as hydrogen peroxide. This oxidative degradation isenhanced by transition metal cations such as iron. It is inhibited byoxygen and free radical scavengers such as ascorbate or propyl gallate.

Acid hydrolysis can be used to reduce the molecular weight of polymers.Also, acid hydrolysis can be used in chemical analysis ofpolysaccharides to break them down to their constituent sugars. Incertain embodiments, although many different acids may be used,generally weak acids are easier to work with than strong acids.

Midi-GAGR and mini-GAGR may be generated by enzymatic biosynthesis usingnucleoside diphosphate glycosyl derivative (e.g., uridine/thymidinediphosphate [UDP/TDP] glycosyl derivative) from a nucleotidetriphosphate (e.g., uridine/thymidine triphosphate) and a glycosylphosphate ester (e.g., glucose-1-phosphate, rhamnose-1-phosphate,glucuronic acid-1-phosphate). UDP/TDP-rhamnose may be generated fromUDP/TDP-glucose by chemical/enzymatic reaction (e.g., epimerization).UDP/TDP-glucuronic acid may be generated from UDP/TDP-glucose bychemical/enzymatic reaction.

FIG. 2 shows the readout of the rheometer measurement for LA-GAGR. Theundigested LA-GAGR had an average molecular weight of approximately99,636 g/mol, which is close to the reported average of 99,600 g/mol.

After 24 hours of enzymatic digestion, the average molecular weight ofthe LA-GAGR digestion product was reduced to approximately 30,245 g/mol(FIG. 3). The three peaks shown in FIG. 3 indicate that α1→3 glucosidasebreaks down LA-GAGR in multiple steps. The 48-hour digestion of LA-GAGR,midi-GAGR, yielded a product of approximately 4,775 g/mol (FIG. 4).

The single narrow peak shown in FIG. 4 indicates that 48-hour enzymaticdigestion yielded a polysaccharide product of an approximate equivalentlength (molecular weight). 72-hour enzymatic digestion of LA-GAGRyielded a product of approximately 718 g/mol (mini-GAGR), which is closeto the molecular weight of the basic repeating sugar units of LA-GAGR(FIG. 5).

Example 2 Mini-GAGR as a Novel Neurotrophic Agent

midi-GAGR protects cortical neurons from the neurocytotoxicity ofamyloid β peptide.

Primary cultures of mouse embryonic cortical neurons (embryonic days of17 [E17], 14 days in vitro [DIV14]) were treated with mock (H₂O) or 1 μMof dextran, alginate, high acyl gellan gum (HA-GAGR), or midi-GAGR for 6h and then with 10 μM Aβ₄₂ for 48 h.

The viability of neurons was assed using LIVE/DEADViability/Cytotoxicity Assay Kit (Invitrogen Inc.). midi-GAGR increasedthe percent of cell survival in neurons exposed to 10 μM Aβ₄₂ by ˜2.2fold (FIGS. 6A-6C). The other sugar polymers had no effect.

Midi-GAGR is a Strong Antioxidant

The antioxidant capacity of midi-GAGR was measured using ABTSAntioxidant Assay Kit (Zenbio Co.). Midi-GAGR showed strong anti-oxidantcapacities (10 μM midi-GAGR=16.9 μM trolox). This was slightly strongerthan antioxidant capacity of LA-GAGR (10 μM LA-GAGR=15.4 μM trolox)(Table 2).

TABLE 2 Antioxidant Capacity of LA-GAGR and midi-GAGR ABTS assay μMTrolox LA-GAGR 15.4 midi-GAGR 16.9

Midi-GAGR has Neuroprotective Effect on Differentiated PC12 Cells andPrimary Neurons

Differentiated dopaminergic PC12 cells and primary mouse embryoniccortical neurons (E17, DIV14) were treated with mock (H₂O) or 1 μM ofdextran, alginate, HA-GAGR, or midi-GAGR for 6 h and insulated byoxidative stressors (30 μM 6-OHDA [PC12] and 10 μM amyloid β peptide[cortical neurons]) for 24 h. The viability of neurons was assessedusing LIVE/DEAD Viability/Cytotoxicity Assay Kit (Invitrogen). midi-GAGRprevented 6-OHDA-induced cell death in differentiated PC12 cells whilemock, dextran, and alginate did not (FIGS. 7A-7C). midi-GAGR alsoincreased the percent of live neurons exposed to amyloid β peptide by˜2.2 fold while other sugars did not (FIGS. 8A-8C). This shows thatmidi-GAGR has neuroprotective effect on both dopaminergic PC12 cells andprimary neurons.

Treatment with midi-GAGR increased the levels of phosphorylated CREB inthe nuclei of cortical neurons.

Differentiated PC12 cells and mouse embryonic cortical neurons (E17,DIV14) were treated with mock, dextran, alginate, HA-GAGR, or midi-GAGR(1 μM) for 24 h and fixed in 3.7% paraformaldehyde. The fixed cells andneurons were immunostained with antibodies against α-tubulin andphosopho-CREB (p-CREB) along with DAPI (nucleus). 1 μM midi-GAGRincreased the levels of nuclear p-CREB by ˜3 fold in PC12 cells (FIGS.9A-9B) and by ˜1.7 fold in cortical neurons (FIGS. 10A-10C), whilenuclear levels of nuclear p-CREB were not significantly increased bymock, dextran, alginate, or HA-GAGR. These data show that midi-GAGRactivates a neurotrophic signaling pathway.

Midi-GAGR has a Neuritogenic Activity

E16 rat embryonic cortical neurons (DIV14) were treated for 2 days withmock (FIG. 11A), 1 μM dextran, or 1 μM midi-GAGR (FIG. 11B), fixed andimmunostained with anti-synaptophysin antibody (green) andrhodamine-labeled phalloidin (actin: red). Scholl ring analysis wasperformed to quantify dendritic arbors—the number of crossings as afunction of distance from the soma between 0 and 120 μm (mean±S.E.M.).The average number of synaptic clusters per 10 μM of distal dendrite wasalso quantified. The number of crossings (0-120 μm) was higher inneurons treated with 1 μM midi-GAGR than mock- and dextran-treatedneurons (FIG. 12A). The density of synaptic clusters was increased by˜20% (p<0.03) in neurons treated with 1 μM midi-GAGR (FIG. 12B).

Neurons Treated with Midi-GAGR Maintain their Neurites

Rat embryonic cortical neurons were treated with mock (FIG. 11C), 1 μMHA-GAGR (high acyl gellan gum), 1 μM dextran, 100 μM ascorbate, or 1 μMmidi-GAGR (FIG. 13A) for 2 days and then with 25 μM 4-hydroxynonenal(4-HNE), a lipid peroxide, for 1 day. After fixation, neurons wereimmunostained with anti-synaptophysin antibody (green) andrhodamine-phalloidin (actin: red). Scholl ring analysis was performed.Neurons pre-treated with mock, dextran, or ascorbate lost neuritis(neuritic atrophy) in the presence of 4-HNE. Conversely, neuronspretreated with 1 μM HA-GAGR prior to 25 μM 4-HNE formed neuritis, butless branched. 25 μM 4-HNE did not cause severe neuritic atrophy inneurons pre-treated with 1 μM midi-GAGR (FIG. 13B). This resultindicates that midi-GAGR prevents neuritic atrophy caused by 4-HNE.

Midi-GAGR Enhances Neurito Genesis and Actin Filopodia Formation inDifferentiated neuro2A (N2A) Cells

N2A cells were starved in serum-free medium for 3 days with mock (FIG.14A) or 1 μM midi-GAGR (FIG. 14B), fixed, and immunostained withanti-α-tubulin antibody (green) and rhodamine-phalloidin (actin: red).The number and total length of neurites in differentiated N2A cells weremeasured. The number and total length of neurites in midi-GAGR-treatedN2A cells were ˜1.7 fold higher than mock-treated cells. midi-GAGR alsoincreased formation of actin filopodia along neurites (FIG. 14D) whilemock did not (FIG. 14C).

Following differentiation for 3 days with mock or 1 μM midi-GAGR, N2Acells were added with 10% FBS plus mock (FIG. 14E), 4-HNE (25 or 50 μM)or H₂O₂ (100 or 200 μM) (FIG. 14F: 200 μM H₂O₂) for 1 day. Cells werethen fixed and immunostained with anti-α-tubulin antibody (green) andphalloidin (actin: red). The total neurite lengths in N2A cells weremeasured using Metamorph.

Midi-GAGR-Treated N2A Cells Form Neurites in the Presence of 25 μM 4-HNEand 200 μM H₂O₂

Midi-GAGR treated N2A cells form neurites in the presence of 25 μM 4-HNEand 200 μM H₂O₂ (FIG. 15A). Depicted is a bar graph showing the numbersof neurites in N2A cells starved in serum-free-medium plus mock or 1 μMmidi-GAGR for 3 days (n=90, 30 cells×3 independent experiments,Mean±SEM, *=p<0.001).

Midi-GAGR treated 4-HNE cells form neurites in the presence of 25 μM4-HNE and 200 μM H₂O₂ (FIG. 15B). Depicted is a bar graph showing theaverage total neurite lengths of N2A cells starved in serum-free mediumplus mock or 1 μM midi-GAGR for 3 days and then treated with mock, 100or 200 μM H₂O₂, or 25 or 50 μM 4-HNE (n=90, 30 cells×3 independentexperiments, mean±SEM, * (blue star)=p<0.001 compared to mock, * (redstar)=p<0.001 compared to treatment with free radicals minus midi-GAGR).

The average total neurite length in mock-treated cells was decreased to˜50% of mock treatment by 4-HNE (25 μM) and to ˜1% or mock by H₂O₂ (200μM) while cells pretreated with 1 μM midi-GAGR maintained their neuritesin the presence of 25 μM 4-HNE and 200 μM H₂O₂ (FIG. 15B). These dataindicate that midi-GAGR prevents neurite atrophy caused by 4-HNE andH₂O₂.

Midi-GAGR enhances total neurite length of primary cortical neurons(FIG. 16). Midi-GAGR enhances total neurite length of primary corticalneurons. Depicted is a bar graph showing the average total neuritelength (μM) of primary cortical neurons treated with mock or midi-GAGR,low acyl gellan gum (LA), high acyl gellan gum (HA), dextran, oralginate (mean±SEM).

Midi-GAGR Penetrates an In Vitro BBB Layer

An in vitro blood-brain barrier (BBB) filter system was generated thatconsisted of bEND3 cells and astrocytes, as shown in the schematicillustration in FIG. 17. The filter system was placed on the top of eachwell of a 24-well plate, under which N2A cells were seeded on coverslipsand incubated in serum-free DMEM. Mock, 1 or 10 μM midi-GAGR (mGAGR) wasinjected onto the top of the filter system. After 3 daysdifferentiation, the filter systems were removed and N2A cells weretreated with mock or 25 μM 4-HNE for 1 day. N2A cells that were underthe filters into which 1 or 10 μM midi-GAGR was injected showed longerneurites that those injected with mock (FIG. 18). N2A cells that wereunder the filters into which 1 or 10 μM midi-GAGR was injected from longneurites even in the presence of 25 μM 4-HNE while cells injected withmock did not. This shows that that midi-GAGR can penetrate the in vitroBBB filter system.

Midi-GAGR Binds to NCAM-180

A midi-GAGR-epoxy-sepharose 6B column (GE Healthcare Biosciences,Pittsburgh, Pa.) was used to purify midi-GAGR-interacting protein(s)from the plasma membrane fraction of mouse brain synaptosomes. Multiplepurification experiments yielded the same result, showing the presenceof a ˜180 kD protein only in the eluate from midi-GAGR beads. Massspectrometry analysis showed that the 180 kD protein was NCAM-180.Activation of NCAM and FGFR induces MAPK pathways, resulting inactivation of CREB. midi-GAGR thus exerts its neurotrophic effect viaits interaction with NCAM-180.

FIG. 19 shows that Midi-GAGR binds to NCAM1. Depicted is a bar graphshowing the normalized spectrum count for mock (control) or midi-GAGRbinding to neurexin, carboxypeptide D or NCAM1. Data is the result oftwo independent experiments.

Example 3 Enhancement of Osteogenesis of Adult Mesenchymal Stem Cells byMini- and Midi-GAGR

Human bone marrow-derived mesenchymal stem cells were cultured in eitherminimum osteogenic media or α MEM media on 24 well plates for 14 days.Cells were treated with control, 0.1 μM mini-GAGR, 1.0 μM mini-GAGR, 0.1μM midi-GAGR, or 1.0 μM midi-GAGR every two days. After 14 days, cellswere stained with Alizarin S and imaged using a light phase microscope.

Both mini- and midi-GAGR enhanced bone cell formation from human adultmesenchymal stem cells in the minimum osteogenic medium at very lowconcentrations (0.1 μM and 1.0 μM) (FIGS. 20A-20B and FIGS. 21A-21B)mini- and midi-GAGR enhanced bone cell formation ˜500 fold compared tocontrol minimum osteogenic media alone. Neither mini- nor midi-GAGRinduced or enhanced osteogenesis in α MEM (non-osteogenic condition)(FIGS. 20A-20B and FIGS. 21A-21B).

Both mini- and midi-GAGR enhanced bone cell formation from adultmesenchymal stem cells in the osteogenic micro-environment found withinbone. The osteogenic effect of mini- and midi-GAGR is limited to bone,and has no osteogenic effect in other intra-body regions.

Neither mini- nor midi-GAGR enhanced the expansion of stem cells, whichexcludes the possible implication of tumorigenesis of stem cells bymini- and/or midi-GAGR, a major setback for stem-cell based therapeuticapproaches.

Example 4 BBB-Permeable, Neuroprotective and NeurotrophicPolysaccharide, Midi-GAGR Materials and Methods

In order to increase water solubility and diffuse-ability, LA-GAGR wascleaved into smaller sizes by enzymatic digestion (α[1→3] glycosidase)for 24, 48, and 72 h. The MWs of its cleavage products were determinedusing Parallel Plate Rheometer that measures shear storage modulus andloss modulus and yields the viscosity profile of polysaccharides. Fromthe viscosity-storage modulus profiles, the MW distributions of thecleavage products were determined using RheoAnalyzer program. Thevalidity of the RheoAnalyzer program was verified by running polystyrenestandard (NBS 706) on the Parallel Plate Rheometer and determining MWfrom the viscosity profile. The average MW of LA-GAGR was ˜99,639g/mole. The MWs of 24-h, 48-h, and 72-h cleavage products were ˜30,245g/mole, ˜4,775 g/mole, and ˜718 g/mole, respectively. The average MW of˜4,775 g/mole (named ‘midi-GAGR’) is equivalent to six repeating unitsand that of ˜718 g/mole (named ‘mini-GAGR’) is to one repeating unit.Two small-size LA-GAGR products, midi-GAGR and mini-GAGR, were chosenfor further examination regarding their neuroprotective effect.

Embryos (at the embryonic day of 17 [E17]) from female pregnant mice(BALB/C, Charles River Laboratories International Inc., Wilmington,Mass.) and female pregnant rats (E17, Sprague Dawley [SD],bred-in-house) were used to isolate primary cortical neurons for invitro primary culturing. Adult female SD rats (12-16 weeks old,bred-in-house) were used for in vivo studies to examine theBBB-permeability and neurotrophic effect of midi-GAGR. 12-month-old3×Tg-AD mice (female, B6; 129-Psen1tm1Mpm Tg [APPSwe, tauP301L]1Lfa/Mmjax, Jackson Laboratory, Bar Harbor, Me.) were used for thestudies to examine the effects of midi-GAGR on neurotrophic andneurodegenerative markers in 3×Tg-AD mouse brains. Animals were housedat room temperature under a 12-h light/dark cycle. Food and water wereprovided ad libitum. All experiments were performed during the lightphase (7 am-7 pm). All the procedures of animal use described in thisstudy were approved by the Institutional Animal Care and Use Committee(IACUC) of University of Toledo College of Medicine and Life Science inaccordance with National Institutes of Health guidelines.

Antibodies

Antibodies to FGFR1 (SAB4300488), neurofilament 200 (NF200, N4142), andα-tubulin (T9026), synaptophysin (S5768), and βIII-tubulin (T2200) werepurchased from Sigma (St. Louis, Mo.). Antibodies to PSD95 (sc-32290),pCREB (P-Ser133, sc-7978), CREB (sc-377154), glyceraldehyde-3-phosphatedehydrogenase (GAPDH, sc-32233), and growth associated protein 43(GAP-43, sc-17790) were purchased from Santa Cruz Biotechnology, Inc.(Santa Cruz, Calif.). Antibody to PHF-tau (P-Ser202, AT8, MN1020B) waspurchased from Thermo Scientific (Rockford, Ill.). Antibodies to Iba1(ab5076) and pCREB (P-Ser133, ab30651) were purchased from Abcam Inc.(Cambridge, Mass.). Antibody to tau (610672) was purchased from BDTransduction Laboratories (Lexington, Ky.).

Hydrolysis of Low Acyl Gellan Gum

1 mL of 1% salicin solution (1 g salicin [Sigma] in 100 mL of 0.1 Macetate buffer [pH 5]) was pre-warmed at 37° C. for 6-8 minutes andmixed with 2 mg of α(1→3)-glucosidase (Sigma) to make the enzymesolution for the hydrolysis of low acyl gellan gum (LA-GAGR, CPKelco Co.[Atlanta, Ga.]). The enzyme solution was diluted to 0.1 mg/mL beforeuse. 0.48 g of LA-GAGR was dissolved in 80 mL of the acetate/salicinsolution. 8 mL of LA-GAGR solution was mixed with 2 mL of the enzymesolution in 15 mL polypropylene comical tubes and incubated at 37° C.(80 rpm) for 24, 48, or 72 h for the enzymatic digestion of LA-GAGR.Enzyme reaction was stopped by incubation in a hot water bath for 5 minand dried in a vacuum dryer (−60 cm Hg gauge) at 70° C. Dried gel pelletwas then washed extensively in de-ionized water by stirring for 48 h(fresh water replaced every 12 h) to wash off salts and enzyme from thepellet and processed for viscosity measurement using a parallel platerheometer (PPR, Rheometrics, Inc., Piscataway, N.J.) equipped withrheometer software, TA Orchestrator (TA Instrument, Inc., New Castle,Del.). From the viscosity-storage modulus profiles, the MW distributionsof the cleavage products were determined using the RheoAnalyzer program(TomCoat Oy, Inc., Finland). The pellet was dissolved in 100 mLde-ionized water, autoclaved at 120° C. for 45 min, aliquoted, and keptat −80° C. until use.

Drug Treatment of Neuro2A (N2A) Cells

N2A cells (passage 7-10 [ATCC® CCL-131™]) were sparsely seeded oncoverslips in Dulbecco's modified Eagle's medium (DMEM, LifeTechnologies, Grand Island, N.Y.) containing 10% heat-inactivated fetalbovine serum (FBS), 5 g/L D-glucose, 110 mg/L sodium pyruvate, and 1×PenStrep (Life Technologies) and incubated at 37° C. in a humidified 5% CO₂incubator for 48 h. Then, serum-containing medium was replaced withserum-free medium containing vehicle (H₂O) or different concentrationsof midi-GAGR (0.001, 0.01, 0.1, 1, and 10 μM) and incubated for 3 daysprior to immunocytochemistry using anti-α-tubulin antibody followed bysecondary Alexa_(488nm) antibody (Life Technologies). Coverslips werethen mounted on glass slides using Fluoromount G (Fisher Scientific,Pittsburgh, Pa.). The images of cells were taken using a TCS SP5multi-photon laser scanning confocal microscope (Leica Microsystems,Bannockburn, Ill.). The confocal microscope is equipped withconventional solid state, a ti-sapphire tunable multi-photon laser(Coherent, Santa Clara, Calif.), and acousto optical beam splitter AOBS.Images were taken with either 40× or 20×Zeiss alpha plan fluor oilobjective (1.4 NA). Cells having the neurite length longer than 2 timesthe diameter of cell body were chosen for image analysis. To examine theprotective effect of midi-GAGR against oxidative stress-induced neuriteatrophy, differentiated N2A cells were pre-treated with midi-GAGR (0,0.001, 0.01, 0.1, 1, and 10 μM) and then with either 4-hydroxynonenal(4HNE) (Cayman Chemical, Ann Arbor, Mich.) or H₂O₂ (Sigma). First, thetoxic dose ranges of 4HNE and H₂O₂ that cause neurite atrophy weredetermined by treating differentiated N2A cells with 0, 1, 5, 10, or 25μM of 4HNE for 48 h or 0, 1, 10, 50, 100, or 200 μM of H₂O₂ for 24 h.Then, the doses that showed maximum inhibitory effects were used totreat differentiated N2A cells along with 0, 0.001, 0.01, 0.1, 1, and 10μM of midi-GAGR. The total neurite lengths of N2A cells in differentconditions were measured using ‘Metamorph’ software (Molecular Devices,Sunnyvale, Calif.) and used to calculate average total neurite lengths.

Drug Treatment of Primary Rodent Cortical Neurons

The protective effect of polysaccharides on primary cortical neuronsfrom 4HNE, H₂O₂, and Aβ₄₂ peptide (Sigma) was examined using LIVE/DEAD®Viability/Cytotoxicity Assay Kit (Life Technologies). Female pregnantmice (BALB/C, E17, Charles River Laboratories International, Inc.) orfemale pregnant SD rats (E17, bred in house) were anesthetized anddissected to obtain 8-9 embryos per animal. Cortical neurons wereisolated from embryonic brains and differentiated onpoly-L-lysine-coated coverslips in B27/neurobasal medium. For drugtreatment before free radical treatment, mouse cortical neurons (5 daysin vitro [DIV5]) were treated with vehicle (H₂O), 1 μM of 5 kD dextran(Sigma), alginate (Sigma), midi-GAGR, 0.01 μM of high acyl gellan gum(HA-GAGR, CPKelco Co.), or 0.1 μM of LA-GAGR for 24 h and then treatedwith 10 μM 4HNE or 50 μM H₂O₂ for 24 h, or 2 μM Aβ₄₂ peptide for 48 hprior to viability/cytotoxicity assay. For drug co-treatment with freeradicals, rat cortical neurons (DIV5) were treated with 1 μM of dextran,alginate, or midi-GAGR, 0.01 μM of HA-GAGR, or 0.1 μM of LA-GAGR alongwith either 10 μM 4HNE (24 h) or 2 μM Aβ₄₂ peptide (48 h). To examinethe extent to which midi-GAGR-mediated neuroprotection depends on FGFR1,rat cortical neurons (E17, DIV5) were pre-treated with 4 μM SU5402(Sigma) for 6 h and treated with 10 μM 4HNE and either vehicle or 1 μMmidi-GAGR for 24 h prior to cell viability/cytotoxicity assay. Ascontrols, neurons were treated with SU5402, midi-GAGR, 4HNE, 4HNE plusmidi-GAGR, or SU5402 plus midi-GAGR.

For cell viability/cytotoxicity measurement, neurons on glass coverslipswere incubated in 1×PBS containing 2 μM calcein AM (live cells: green)and 4 μM ethidium homodimer-1 (dead cells: red) for 10 min at 37° C.Immediately thereafter, neurons were imaged by 10×objective using afluorescence Olympus IX71 microscope (Olympus America Inc., CenterValley, Pa.) and, for the acquisition of high-quality images, using TCSSP5 multi-photon laser scanning confocal microscope.

Co-Culturing of Microglial Cells and Primary Rat Cortical Neurons

Microglial cell culture was prepared from the whole brain tissues(except of the cerebellum) of rat pups at postnatal day 3 (P3). Briefly,whole brain tissues except of the cerebellum were dissected andre-suspended in L-15 media on ice. Brain tissues were centrifuged at1,000×g for 3 min at 4° C. After the supernatant over brain tissuepellet was removed, the pellet was re-suspended in fresh L-15 media,followed by mechanical digestion using pasteur pipette. After thedigestion, the resuspension was filtered through cell strainer (porediameter=70 μm). The flow-through was centrifuged at 1,000×g for 3 minat 4° C. Then, cell pellet was re-suspended in DMEM media containing 10%FBS and 1× penicillin/streptomycin and plated at a high density in T75culture flasks. The medium was exchanged with fresh medium every fourdays. After 8-10 days, culture flask caps were covered with parafilm toprevent gas exchange with environmental air. Flasks were then shaken inan orbital shaker at 220 rpm for 4 h at 37° C. Media was then collectedinto a conical tube and centrifuged at 800×g for 10 min. The resultingcell pellet (mostly microglial cells) was then re-suspended in aneurobasal media containing B27 supplement and plated in filter insert(0.4-μm pore diameter) at a density of 2×10⁵ cells. The filter insertscontaining microglial cells were transferred to a 24 well platecontaining rat cortical neuron cultures (DIV6) at the bottom of eachwell. Microglial cells were treated with either 2 μM Aβ₄₂ peptide orvehicle (H₂O) and neurons with either 1 μM midi-GAGR or vehicle (H₂O)for 48 h prior to live-dead assay. In addition, whether microglial cellspenetrated the filter and fell down to neurons at the bottom of well ornot were examined by staining the cells on coverslips at the bottom ofwell by staining the coverslips with anti-Iba1 antibody forimmunocytochemistry and confocal microscopy.

Analysis of Neurite Outgrowth and pCREB in Primary Mouse CorticalNeurons

To analyze the effect of polysaccharides on neurite outgrowth, primarymouse cortical neurons were treated with vehicle (H₂O) or 1 μM ofdextran, alginate, or midi-GAGR, 0.01 μM of HA-GAGR, or 0.1 μM ofLA-GAGR for 2 days prior to immunocytochemistry using anti-α-tubulinantibody and secondary Alexa_(488nm) antibody. An etched grid coverslipcontaining 200 numbered boxes was used to select neurons objectively forimage acquisition and analysis. A total of 24 boxes were randomlyselected per treatment group. All the neurons having total neuritelength longer than 4 times of the diameter of neuron cell body werechosen for image analysis. The total length of the neurites of eachneuron was measured using Metamorph and used to calculate average totalneurite length. To examine the effect of polysaccharides on thephosphorylation (activation) of nuclear CREB, neurons were stained withanti-pCREB antibody (Abcam & Santa Cruz biotechnologies, 2nd antibodywith Alexa_(568nm)), anti-α-tubulin antibody (Sigma, 2nd antibody withAlexa_(488nm)), and DAPI (Sigma) after two-day incubation with vehicle(H₂O) or 1 μM of dextran, alginate, or midi-GAGR, 0.01 μM of HA-GAGR, or0.1 μM of LA-GAGR. Then, to identify midi-GAGR-induced signaling pathwaythat induces CREB phosphorylation, primary mouse cortical neurons werepre-treated with the inhibitors of FGFR1 (SU5402 [Santa CruzBiotechnologies], 4 μM), PKC (staurosporine [Sigma], 3 nM), MEK (U0126[Sigma], 10 μM), PI3K (LY294002 [Sigma], 20 μM), CaMKII (KN-62[Calbiochem, Billerica, Mass.], 10 μM), or FAK (PF-573228 [Sigma], 1 μM)for 6 h and then with 1 μM midi-GAGR for 48 h prior to the staining ofpCREB and α-tubulin. The images of neurons on coverslips were taken byconfocal microscopy at the same gain (850), offset (−0.01), and exposuretime (2 sec). The intensity of the staining of nuclear pCREB wasmeasured using Metamorph and used to calculate average intensity. Inaddition, the phosphorylation of CREB in the cytosols of mouse corticalneurons treated with polysaccharides were detected by immunoblotting.Primary mouse neurons were dissected from 16 mouse embryos (E17) andplated in the wells of 6-well plates (1×10⁶ cells/well), differentiatedfor 6 days, and treated with polysaccharides for 48 h. Then, neuronswere harvested and lysed in 1% Igepal CA-630 (Sigma)-containing PMEEbuffer plus protease and phosphatase inhibitor cocktails (Sigma) forprotein extraction. Extracted proteins were separated in 4-12% NuPAGEBis-Tris protein gels (Life Technologies) and transferred tonitrocellulose membrane (GE Healthcare Life Science, Pittsburgh, Pa.)using a semidry blotter (Hoefer, Inc., San Francisco, Calif.). Theprotein bands on blots recognized by anti-pCREB (Santa CruzBiotechnologies) and anti-CREB antibodies were detected on AmershamHyperfilm™ ECL films (GE Healthcare Life Science) using SuperSignal®West Pico Chemiluminescent Substrate (Thermo Scientific).

Examination of the In Vivo Neurotrophic Effect of Midi-GAGR

40 μL of 1 mM midi-GAGR or sterile H₂O (vehicle) was administeredintranasally into the nostrils (20 μL/nostril) of SD rats (4 rats pereach) using a pipette. Animals were kept in anesthetized condition using4% isoflurane and at supine position during administration to preventthe squirting-out of drug. Animals were sacrificed by decapitation at 6h, 24 h, or 48 h after the administration. Whole brain wasmicro-dissected into the frontal cortex, hippocampus, and the rest ofbrain. Then, tissues were homogenized in the 2-fold volume of 1×PMEEbuffer containing 1% Igepal CA-630 and protease inhibitor cocktail usinga 2-mL Teflon homogenizer. The homogenization was then incubated on icefor 30 min at 4° C., followed by centrifugation at 14,500×g for 30 min.The supernatant was collected and its protein concentration was measuredby Bradford assay. 20 μg of proteins was loaded onto each well of NuPage4-11% Bis-Tris protein gels. Immunoblotting was performed using theantibodies to NF200, GAP-43, and GAPDH. The densities of protein bandswere measured using Image J and normalized to that of the loadingcontrol, GAPDH. Normalized values were used to calculate averagenormalized band densities.

Examination of the Interaction of Midi-GAGR with FGFR1 in BrainSynaptosomal Plasma Membrane

To examine whether midi-GAGR interacts with synaptosomal FGFR1 or not,an affinity chromatography using midi-GAGR-conjugated sepharose beadswas performed. Either midi-GAGR or dextran were conjugated toepoxy-activated sepharose 6B that is a pre-activated medium that can beconjugated to the hydroxyl groups of carbohydrates. Briefly, 200 μL of7.4 mM midi-GAGR or 5 kD dextran in H₂O was mixed with 200 μL ofepoxy-activated sepharose beads in a microtube and incubated on arotator (16 h, 37° C.). The mixture was spun down at 1,000×g (10 min) toseparate bead-bound polysaccharides from unbound. Unoccupied activesites on beads were blocked by incubation (4 h, 45° C.) in 1 Methanolamine (pH 8). Then, beads were washed with three cycles ofalternating pH solutions—0.1 M acetate buffer (pH 4) and 0.1 M Tris-HClbuffer (pH 8), both containing 0.5 M NaCl. The conjugation ofpolysaccharides to beads was confirmed by phenol-sulfuric acidcolorimetry. Cerebral cortices were dissected from four female mice(BALB/C, 8 wks old) and homogenized with a hand grinder in 1 mL of PMEEhomogenization buffer plus 1% Igepal CA-630 and protease inhibitors(PIs). The homogenate was centrifuged at 1,000×g (10 min) to removenuclei and undisrupted cells. The supernatant was subjected to 5-6strokes through 27G needle. Post-nuclear supernatant was centrifuged at1,000×g (15 min). The supernatant was diluted to 1:2 with IgepalCA-630-free PMEE buffer (to make 0.5% Igepal CA-630) and stored at −80°C. until use. Then, the supernatant was incubated with 100 μL of eithermidi-GAGR-conjugated or dextran-conjugated beads. After 24-h incubationon a rotator at 4° C., the beads were washed three times with 0.5 mL ofPMEE buffer to remove proteins that nonspecifically bind to beads. Then,the beads were boiled for protein elution in a SDS loading buffer.Eluted proteins were separated in a SDS-NuPAGE gel and processed forimmunoblotting using FGFR1 antibody.

Examination of the Effects of Midi-GAGR on Neuronal Activity andNeurodegenerative Markers in 3×Tg AD Mice

12-week-old 3×Tg-AD mice (female, ˜20 g, B6.Cg-Psen1tm1Mpm Tg [APPSwe,tauP301L]1Lfa/J) were used to examine the efficacy of midi-GAGR inrestoring neuronal activity and reducing neurodegeneration in AD mousebrains. Three 3×Tg AD mice were intranasally administered with eithersterile H₂O (vehicle) or 7.4 mM midi-GAGR (40 μL total, 20 μL/nostril)every day for 14 days after 4% isoflurane anesthetization and thensacrificed by decapitation. Whole brain was micro-dissected to obtainthe cortices and hippocampi. The tissues were homogenized in the 2-foldvolume of 1×PMEE buffer containing 1% Igepal CA-630 plus phosphatase andprotease inhibitor cocktail using a miniature cell grinder for 1.5 mLmicrotube. The homogenization was then incubated on a rotator for 30 minat 4° C., followed by centrifugation at 14,500×g for 30 min. Thesupernatant was collected and its protein concentration was measured byBradford assay. 30 μg of proteins was loaded onto NuPage 4-11% Bis-Trisprotein gels. Immunoblotting was performed using the antibodies toNF200, GAP-43, PSD95, synaptophysin, pCREB, CREB, p-tau (AT8), tau, andGAPDH. The densities of protein bands were measured using Image J andnormalized to that of the loading control, GAPDH. Normalized values wereused to calculate average normalized band densities.

Statistical Analysis

All cell culture experiments were replicated multiple times withdifferent batches of cell cultures. Microscopic analysis was performedblindly by students. Statistical significance between two groups wascalculated using unpaired student's t-test with a value of p<0.05 thatwas considered statistically significant. Multiple comparisons wereperformed using one-way ANOVA followed by Dunnett's or Bonferroni'smultiple comparisons tests (GraphPad Prism software, La Jolla, Calif.).

Generation of Cleavage Products from LA-GAGR

LA-GAGR was cleaved into smaller sizes by enzymatic digestion(α(1→3)-glycosidase) for 24, 48, and 72 h. The MWs of LA-GAGR's cleavageproducts were determined using Parallel Plate Rheometer that measuresshear storage modulus and loss modulus and yields the viscosity profileof polysaccharides. From the viscosity-storage modulus profiles, the MWdistributions of the cleavage products were determined usingRheoAnalyzer program. The validity of the RheoAnalyzer program wasverified by running polystyrene standard (NBS 706) on the Parallel PlateRheometer and determining MW from the viscosity profile.

The average MW of LA-GAGR was ˜99,639 g/mole, which is close to thevalue reported by CPKelco Co. The MWs of 24-h, 48-h, and 72-h cleavageproducts were ˜30,245 g/mole, ˜4,775 g/mole, and ˜718 g/mole,respectively. The average MW of ˜4,775 g/mole (named ‘midi-GAGR’) isequivalent to six repeating units and that of ˜718 g/mole (named‘mini-GAGR’) is equivalent to one repeating unit.

Results for Example 4 Midi-GAGR Rescues Neurites from the Atrophy Causedby 4HNE and H₂O₂

Differentiated N2A cells were used to examine if midi-GAGR and mini-GAGRprotect neurites from the oxidative insults of 4HNE and H₂O₂ as LA-GAGRdoes. The atrophic dose ranges of the radicals were determined bytreating differentiated N2A cells with increasing concentrations of 4HNEand H₂O₂ for 48 h and 24 h, respectively. Treated cells were fixed,immunostained by anti-α-tubulin antibody, imaged by confocal microscopy,and examined regarding total neurite length. The average total neuritelength of N2A cells was decreased in a dose-dependent manner in responseto 4HNE and H₂O₂ to maximum extents at 25 μM 4HNE (FIG. 22B, FIG. 22D)and 200 μM H₂O₂ (FIG. 22C, FIG. 22E) compared to vehicle (FIG. 22A).

25 μM and 200 μM were the maximum doses of 4HNE and H₂O₂, respectively,that caused almost complete neurite atrophy in differentiated N2A cells.Differentiated N2A cells were treated with increasing concentrations ofmidi-GAGR prior to the treatment with either 25 μM 4HNE or 200 μM H₂O₂.Treatment with 0.1 and 1 μM midi-GAGR prior to 4HNE treatment rescuedneurites up to ˜70% of the control level (vehicle) (FIG. 22F, FIG. 22H).

Similarly, treatment with 0.1 and 1 μM midi-GAGR prior to H₂O₂ treatmentrescued neurites up to ˜100% of the control level (FIG. 22G, FIG. 22I).The protective effect of mini-GAGR was examined, but it was found thatmini-GAGR was not as potent as midi-GAGR in protecting neurites from thefree radicals. Therefore, midi-GAGR (which showed strongerneuroprotective effect against 4HNE and H₂O₂) was used in furtherexperiments.

Midi-GAGR Reduces the Apoptosis of Rodent Cortical Neurons Caused by4HNE, H₂O₂, and Amyloid β Peptide

Midi-GAGR attenuated neurite atrophy caused by free reactive radicals.It is now shown herein that midi-GAGR protect neurons from the deathcaused by free radical insults. The extent to which midi-GAGR protectsthe primary culture of rodent cortical neurons from 4HNE and H₂O₂ wasdetermined. In addition to the radicals, amyloid β peptide (Aβ₄₂) wasalso tested. Aβ₄₂ peptide is a major causative factor that causesoxidative stress and neuron death. Mouse cortical neurons (E17, DIV5)were treated with 10 μM 4HNE (24 h), 50 μM H₂O₂ (24 h), or 2 μM Aβ₄₂peptide (48 h) after the treatment of the neurons with vehicle (H₂O),midi-GAGR (1 μM), dextran (1 μM), alginate (1 μM), LA-GAGR (0.1 μM), orHA-GAGR (0.01 μM) for 24 h. The concentrations of 4HNE, H₂O₂, and Aβ₄₂peptide were chosen according to their patho-physiologicalconcentrations. The concentrations of LA-GAGR and HA-GAGR were chosenbecause, within the same volume, the total numbers of sugar units in thepolysaccharides at the concentrations are close to that of 1 μMmidi-GAGR. The viability of neurons was assessed using LIVE/DEAD®Viability/Cytotoxicity Assay Kit in which membrane-permeant calcein AMis cleaved by esterase in live cells, thus yielding green fluorescence,and membrane-impermeant ethidium homodimer-1 stains the nucleic acids ofplasma membrane-compromised cells with red fluorescence. The numbers ofgreen (live) and red (dead) neurons in each condition were counted usingMetamorph. Although the intensities of green fluorescent signals inneurons treated with either free radicals or Aβ₄₂ peptide were weak, theneurons were included in the counting. About 8-9% of vehicle-treatedneurons died during the process of live/dead cell assay (FIGS. 23A-23F,FIGS. 24A-24C).

Upon exposure to 10 μM 4HNE, ˜26% of mouse cortical neurons died (FIG.23B, FIG. 24A) while pre-treatment with 1 μM midi-GAGR (FIG. 23C) and0.1 μM LA-GAGR reduced neuron death to 14% and 10%, respectively (FIG.24A). HA-GAGR and alginate also reduced the percent of neuron deathcaused by 4HNE while dextran did not (FIG. 24A). Exposure to 50 μM H₂O₂caused neuron death in ˜25% of cortical neurons that were pre-treatedwith vehicle (FIG. 23D, FIG. 24B). Pre-treatment with either alginate ordextran did not reduce H₂O₂-caused neuron death (FIG. 24B). Conversely,pre-treatment with midi-GAGR (FIG. 23E), LA-GAGR, or HA-GAGR reducedneuronal death to ˜13% (FIG. 24B). These results show that midi-GAGR,LA-GAGR, and HA-GAGR protects rodent cortical neurons from both H₂O₂ and4HNE while dextran and alginate cannot.

The extent to which midi-GAGR protects cortical neurons from Aβ₄₂peptide was examined. Exposure to 2 μM Aβ₄₂ peptide caused the death ofabout 30% of neurons pre-treated with vehicle (FIG. 23F, FIG. 24C).Pre-treatment with HA-GAGR, alginate, or dextran did not decrease thepercent of neuron death compared to pre-treatment with vehicle (FIG.24C). Conversely, pre-treatment with either midi-GAGR or LA-GAGR reducedneuron death to ˜10%, the control level (FIG. 23G, FIG. 24C). Thus, onlymidi-GAGR and LA-GAGR protects cortical neurons from Aβ₄₂ peptide, whileother polysaccharides cannot.

Midi-GAGR Reduces the Apoptosis of Rodent Cortical Neurons fromCo-Treated 4HNE or Aβ₄₂ Peptide

It was determined whether midi-GAGR protects cortical neurons fromco-treated 4HNE or Aβ₄₂ peptide. Rat cortical neurons (E17, DIV5) weretreated with either 10 μM 4HNE (24 h) or 2 μM Aβ₄₂ peptide (48 h) andeither vehicle or 1 μM midi-GAGR. Then, the viability of neurons wasassessed using LIVE/DEAD® Viability/Cytotoxicity Assay Kit. Exposure to10 μM 4HNE caused apoptosis in ˜27% of vehicle-treated neurons whileco-treatment with midi-GAGR reduced the percent of neuron death to ˜9%(FIG. 25A). Treatment with 2 μM Aβ₄₂ peptide caused death in ˜29% ofvehicle-treated neurons, while co-treatment with midi-GAGR reduced thepercent of neuron death to ˜15% (FIG. 25A). These results show thatmidi-GAGR also protects rodent cortical neurons from co-treated 4HNE orAβ₄₂ peptide.

Midi-GAGR Protects Rodent Cortical Neurons from Microglial CellsActivated by Aβ₄₂ Peptide

It was determined whether midi-GAGR protects primary rodent corticalneurons from microglia cells activated by Aβ₄₂ peptide. Microglial cellswere isolated from rats on the postnatal day 1 and seeded in0.4-μm-pore-size filter insert that fits into the well of 24-well plate.The filter inserts were transferred to a 24-well plate in which primaryrat cortical neurons (E17) were cultured at the bottoms of wells for 6days (DIV6). Then, microglia cells were treated with 2 μM Aβ₄₂ andneurons with either vehicle or 1 μM midi-GAGR. After 48 h, the viabilityof neurons was assessed using LIVE/DEAD Viability/Cytotoxicity AssayKit. Around 25% of the neurons treated with vehicle died under untreatedmicroglial cells (FIG. 25B). Upon treatment of microglial cells withAβ₄₂ peptide, the percent of death in neurons treated with vehicle wasincreased to ˜40%. Conversely, treatment of neurons with 1 μM midi-GAGRreduced death to ˜27% that was close to the percent of dead neuronsunder untreated microglial cells (FIG. 25B). Thus, it is clear thatmidi-GAGR protects rodent cortical neurons from activated microglialcells.

Midi-GAGR Enhances Neurite Outgrowth in N2A Cells and Rodent CorticalNeurons

The neuritogenic effect of midi-GAGR on N2A cells was examined. N2Acells were differentiated with increasing concentrations (0, 0.001,0.01, 0.1, 1, and 10 μM) of midi-GAGR for 48 h. Then, cells were fixedand immunostained with anti-α-tubulin antibody. The total length ofneurites per cell was measured using Metamorph and used to calculateaverage total neurite length per treatment group. At 0.1 and 1 μM, theaverage total neurite length of midi-GAGR-treated N2A cells reached ˜1.7fold higher than that of vehicle-treated cells (FIGS. 26A-26C;438.21±20.55 μm [0.1 μM] and 457.76±41.66 um [1 μM] vs. 257.51±45.16 μm[vehicle], p<0.05). At 10 μM, average total neurite length wasdecreased, which is similar to the pattern of neuritogenesis in cellstreated with FGL. The neuritogenic effect of midi-GAGR on primary mousecortical neurons (E17, DIV4) was examined. Neurons were incubated withvehicle, midi-GAGR (1 μM), dextran (1 μM), alginate (1 μM), LA-GAGR (0.1μM), or HA-GAGR (0.01 μM) for 2 days and immunostained with the antibodyto βIII tubulin (FIGS. 27A-27F). The total neurite length of each neuronwas measured to calculate average total neurite length per condition.Then, average total neurite length per condition was divided by that ofvehicle treatment to obtain fold change in average total neurite lengthper condition. Compared to vehicle treatment (FIG. 27A), midi-GAGR (FIG.27B) and LA-GAGR (FIG. 27C) increased average total neurite length by˜1.6 fold (FIG. 27G). Conversely, HA-GAGR (FIG. 27D), alginate (FIG.27E), and dextran (FIG. 27F) did not enhance neuritogenesis (FIG. 27G).Thus, midi-GAGR and LA-GAGR have strong neuritogenic effect on rodentcortical neurons.

Midi-GAGR Activates CREB, a Neurotrophic Transcriptional Factor

midi-GAGR-treated mouse cortical neurons were stained with the antibodyto pCREB, a marker for activated neurotrophic signaling pathways. Mousecortical neurons (E17, DIV4) were treated with vehicle (FIG. 28A),midi-GAGR (1 μM, FIG. 28B), alginate (1 μM, FIG. 28C), dextran (1 μM,FIG. 28D), LA-GAGR (0.1 μM), or HA-GAGR (0.01 μM) for 48 h, fixed,immunostained with the antibodies to α-tubulin and pCREB along withDAPI. The fluorescence intensity (arbitrary number) of stained pCREB inthe nucleus was measured using Metamorph. Vehicle-treated neurons showedthe basal levels of pCREB in the nuclei (FIG. 28A). Conversely,treatment with either midi-GAGR (FIG. 28B) or LA-GAGR significantlyincreased the level of nuclear pCREB while the other polysaccharides didnot (FIG. 28C, FIG. 28D). Treatment with HA-GAGR slightly increased thelevel of nuclear pCREB. The average intensity of stained pCREB in thenuclei of the neurons was calculated. Treatment with either midi-GAGR orLA-GAGR increased the average intensity of nuclear pCREB by ˜2 foldcompared to vehicle treatment (FIG. 28E). To confirm the result of theoptical measurement of the level of pCREB, immunoblotting was performedto detect the phosphorylation of CREB protein using the cytosolsextracted from mouse cortical neurons treated with vehicle orpolysaccharides. Total CREB was also detected by immunoblotting.Compared to control (vehicle treatment), the levels of pCREB weresignificantly increased in neurons treated with either midi-GAGR orLA-GAGR while those in neurons with either alginate or dextran were not(FIG. 28F). The phosphorylation level of HA-GAGR was also increased.Thus, midi-GAGR and LA-GAGR increase the phosphorylation of CREBsignificantly and HA-GAGR does slightly.

Intranasally Administered Midi-GAGR Penetrates the BBB and Increases theExpression of NF200 and GAP-43 in the Frontal Cortex and Hippocampus

The expression of two neurotrophic protein markers, NF200 and GAP-43,was examined in the brains of rats administered intranasally with 40 μLof either H₂O or 1 mM midi-GAGR. At 6, 24, and 48 h after the intranasaladministration, the frontal cortex, hippocampus, and the rest of thebrain were dissected from the rats and processed for immunoblottingusing the antibodies to NF200, GAP-43, and GAPDH (loading control). Theprotein band densities of NF200 and GAP-43 were measured by densitometry(Image J), normalized by those of GAPDH, and used to calculate averagenormalized values. In the frontal cortex, the expression level of NF200was increased to the level significantly higher than control at 6 h and24 h after the administration (FIG. 29A, FIG. 29B). In the hippocampus,that of NF200 was increased to the level statistically higher thancontrol after 24 h. The expression level of GAP-43 was alsosignificantly increased in the frontal cortex at 6 h and 24 h whileslightly increased in the hippocampus at 24 h (FIG. 29A, FIG. 29C). Therest of the brain did not show an increase in NF200 and GAP-43 afterone-time intranasal administration. These results show that intranasallyadministered midi-GAGR enters the brain and exerts its neurotrophiceffect in the frontal cortex and hippocampus within 24 hpost-administration.

Midi-GAGR Binds to FGFR1 and Activates FGFR1 Signaling Pathway

The interaction of midi-GAGR with FGFR1 was examined by affinitychromatography using midi-GAGR-conjugated sepharose column. Eithermidi-GAGR or dextran was conjugated to epoxy sepharose beads accordingto manufacturer's protocol. Whole mouse brains were homogenized forcytosol extraction in PMEE buffer containing 1% Igepal CA-630 andprotease inhibitor cocktail. Brain cytosols were diluted to 1:2 to make0.5% Igepal buffer prior to the incubation with either dextran- ormidi-GAGR-conjugated sepharose beads on a rotating plate for 16 h at 4°C. Nonspecific bindings were removed by extensive washes in PMEE buffer.Beads were boiled for protein elution. FGFR1 was one of the proteinseluted from midi-GAGR beads but not from dextran beads (FIG. 30A).

Pharmacological agents were used to inhibit signaling moleculesdownstream of FGFR1 that mediate CREB phosphorylation. The fluorescenceintensities of nuclear pCREB in mouse cortical neurons (E17, DIV6)pre-treated for 6 h with the inhibitors of FGFR1 (SU5402 [SU], 4 μM),PKC (staurosporine [Stau], 3 nM), MEK (U0126 [U01], 10 μM), PI3K(LY294002 [LY], 20 μM), or CaMKII (KN-62 [KN], 10 μM); and, then with 1μM midi-GAGR (midi) for 48 h were measured. PF-573228 (PF, 1 μM) thatinhibits FAK which sits at the bottleneck of the signaling pathwaydownstream of NCAM180 was also included to examine whether NCAM180 isinvolved in midi-GAGR-mediated CREB phosphorylation or not. Treatedneurons were stained with the antibodies to βIII tubulin (red) and pCREB(green) for the measurement of the average intensities of nuclear pCREB.Compared to neurons treated with vehicle (FIG. 30B, FIG. 30J), thosewith midi-GAGR (midi) showed a significant increase (˜2.6 fold) in theaverage intensity of pCREB (FIG. 30C, FIG. 30J). Conversely,pre-treatment with the inhibitor of FGFR1 (FIG. 30D, FIG. 30J), PKC(FIG. 30E, FIG. 30J), or MEK (FIG. 30F, FIG. 30J) significantlydecreased the average intensity of pCREB (solid lines, *: p<0.01,n=40-50 neurons). In neurons pre-treated with the inhibitor of eitherPI3K or CaMKII, the average intensity of pCREB was decreased slightlybut still statistically significantly (dotted lines, #: p<0.05, n=40-50neurons). In contrast to the inhibitors, pre-treatment with FAKinhibitor did not decrease the average intensity of pCREB inmidi-GAGR-treated neurons (FIG. 30I, FIG. 30J). These results show thatmidi-GAGR activates FGFR1 and its downstream signaling pathwaysconsisting of PKC, MEK, PI3K, and CaMKII, but not NCAM180-FAK signalingpathway for CREB phosphorylation.

How FGFR1-mediated neurotrophic signaling pathway contributes tomidi-GAGR-mediated neuroprotection against oxidative insult wasexamined. Rat cortical neurons (E17, DIV6) were pre-treated for 6 h withFGFR1 inhibitor (SU5402, 4 μM) and then with 10 μM 4HNE for 24 h priorto LIVE/DEAD Viability/Cytotoxicity Assay. Treatment with 4HNE increasedthe percent of dead cells from ˜20% to ˜60% while co-treatment with 1 μMmidi-GAGR decreased that to ˜30% (FIG. 30K). Treatment with SU5402 aloneor SU5402 plus midi-GAGR did not increase the percent of dead cells(FIG. 30K). Interestingly, pre-treatment with SU5402 significantlyincreased the percent of dead cells up to ˜80% upon the post-treatmentwith 4HNE (FIG. 30K). In neurons pre-treated with SU5402 and thentreated with 4HNE, midi-GAGR could not decrease the percent of deadcells. This result show that FGFR1-mediated signaling pathway plays amajor role in midi-GAGR-mediated neuroprotection against oxidativeinsult.

Intranasally Administered Midi-GAGR Increases Neuronal Activity Markersand Reduces Hyperphosphorylated Tau in 3×Tg-AD Mice

It was determined whether midi-GAGR treatment increases the proteinmarkers of neuronal activity and reduces the neurodegeneration marker,hyperphosphorylated tau (P-Ser202), in AD brain. 12-month-old 3×Tg-ADmice that harbor two familial AD mutations, APP_(swe) and PS1_(M146V),and the tau_(P301L) mutation found in frontotemporal dementia were used.Around 12 months of age, 3×Tg-AD mice show synapse loss, Aβ peptideaccumulation, memory deficit, and tau hyperphosphorylation. Female3×Tg-AD mice were used since females show more obvious cognitive defectsthan males. 40 μL of vehicle (sterile H₂O) or 7.4 mM midi-GAGR wereadministered intranasally into female 3×Tg-AD mice once per day for 14days. During midi-GAGR administration, 3×Tg-AD mice did not show anyabnormal behavior. Of note, 3×Tg-AD mice administered with midi-GAGRmade tight nest every day while those with vehicle made loose nests.After 14-day administration, mice were killed for brain extraction.Extracted brains were dissected to obtain the cortices and hippocampi.The brain tissues were lysed in PMEE buffer containing 1% Igepal CA-630and phosphatase and protease inhibitor cocktails for immunoblottingusing antibodies to NF200, GAP-43, PSD95, synaptophysin (SYN), pCREB,CREB, p-tau (AT8), tau, and GAPDH. The protein band densities ofdetected proteins were normalized by those of GAPDH and used tocalculate average normalized band density for each protein. Compared to3×Tg-AD mice administered with vehicle (Veh.), NF200 was increasedsignificantly in the hippocampus of those with midi-GAGR while notchanged in the cortex (FIG. 31A, FIG. 31B). GAP-43 and PSD95, thepostsynaptic markers for increased synaptic activity, were increasedsignificantly in both cortex and hippocampus of 3×Tg-AD mice treatedwith midi-GAGR compared to control while synaptophysin (SYN) remainedunchanged (FIG. 31A, FIG. 31C, FIG. 31D, FIG. 31E). pCREB, anotherpostsynaptic marker that shows increased neurotrophic signaling, wasalso significantly increased in both cortices and hippocampi of 3×Tg-ADmice treated with midi-GAGR compared to control (FIG. 31A, FIG. 31F).Total CREB was slightly increased in the hippocampus ofmidi-GAGR-treated 3×Tg-AD mice while not changed in the cortex (FIG.31A, FIG. 31G). Surprisingly, hyperphosphorylated tau (P-Ser202) wasdrastically decreased in the hippocampus of 3×Tg-AD mice and slightly intheir cortices (FIG. 31A, FIG. 31H). Total tau was not changed (FIG.31A, FIG. 31I). These results show that intranasally administeredmidi-GAGR not only enhances neuronal and synaptic activities in thecortex and hippocampus but also decreased hyperphosphorylated tau, amajor AD facilitator, in the brains of 3×Tg-AD mice.

DISCUSSION

This Example demonstrates that intranasally administered midi-GAGRpenetrates the BBB and exerts its neurotrophic effect for 24 h after onetime administration. This indicates that midi-GAGR has a goodBBB-permeability and a long plasma half-life of ˜24 h.

In the in vitro experiments, midi-GAGR showed a strong neuroprotectiveeffect. At 0.1-1 μM, midi-GAGR protected the neurites of differentiatedN2A cells up to 70-90% from the atrophy caused by thesupra-physiological concentrations of two free reactive radicals, 4HNEand H₂O₂. At 1 μM, midi-GAGR protected primary rodent cortical neuronsfrom the pathological concentrations of post-/co-treated free radicalsand amyloid β₄₂ peptide. The protective effect of midi-GAGR againstco-existing free radicals and amyloid β₄₂ peptide is clinically relevantas treatment is usually applied to the pre-existing pathologicalconditions like high levels of free radicals and amyloid β₄₂ peptide. Itwas also observed that 1 mM midi-GAGR protected rodent cortical neuronsfrom more vicious neurodegenerative factor, activated microglial cells.Thus, it is clear that midi-GAGR is a strong neuroprotective agent. Incontrast, the control polysaccharides dextran (D-Glc polymer), alginate(D-GlcA polymer), and HA-GAGR (highly acylated LA-GAGR), could notprotect neurons from both free radicals and amyloid β₄₂ peptide. Thus,the neuroprotective effect of midi-GAGR is sequence-specific, which mayconfer midi-GAGR a specific binding to a certain molecule such asreceptor.

In addition to the neuroprotective effect, midi-GAGR showed a strongneurotrophic effect both in vitro and in vivo. In vitro, midi-GAGRgreatly enhanced neuritogenesis in N2A cells at 0.1-1 μM and in rodentcortical neurons at 1 μM. Other control polysaccharides such as dextran,alginate, and HA-GAGR could not enhance neuritogenesis. Similarly, inrodent cortical neurons, midi-GAGR increased the nuclear levels ofactivated pCREB, the transcriptional factor that enhances neuronalactivities for survival and memory, while dextran and alginate did not.HA-GAGR activates CREB to some extent but less than midi-GAGR. A similarneurotrophic effect of midi-GAGR in the brains of rodents that wereintranasally administered with midi-GAGR was observed. In the frontalcortices and hippocampi of midi-GAGR-administered animals, the markersfor neuritogenesis (NF200) and synaptic activity (GAP-43) were increasedwithin 24 h after one-time intranasal spray. Thus, this in vitro and invivo evidence clearly manifest the neurotrophic property of midi-GAGR.

A pulldown experiment and pharmacological inhibitor evaluation wasperformed and found that midi-GAGR interacts with FGFR1 and appears toactivate two FGFR1 signaling pathways, FRS2a-Shc-Grb2-PKC-Raf-MEK andPI3K-Akt/PLCγ-Ca2+-CaMKII pathway. Based on the observation that thelevels of pCREB were reduced more drastically by PKC and MEK inhibitorsthan by PI3K and CaMKII, FRS2a-Shc-Grb2-PKC-Raf-MEK pathway plays a moremajor role than PI3K-Akt/PLCγ-Ca2+-CaMKII pathway for CREB activation.It was also found that FAK is not involved in midi-GAGR-FGFR1 signalingpathway as midi-GAGR-induced CREB phosphorylation was not decreased bythe inhibitor of FAK that sits at the bottleneck of NCAM180 signalingpathway. The selective activation of FGFR1 over NCAM180 is beneficial inthe clinical application of midi-GAGR since the activation of NCAM oftenleads to undesired effects. Both FGF (e.g., FGF2) and FGL have shown agreat efficacy in attenuating neurodegeneration and improve memory in ADmice. Moreover, both FGF2 and FGL reduce AD pathogenic factors.Therefore, midi-GAGR may exert the similar beneficial effects to FGF orFGL while with longer plasma half-life than those peptides.

At the low concentrations below 1 μM, dextran and alginate could notprotect rodent cortical neurons from the pathological concentrations of4HNE and H₂O₂. Moreover, the polysaccharides and HA-GAGR could notprotect the neurons from the relatively low but pathologicalconcentration (2 μM) of Aβ₄₂ peptide. In contrast to the controlpolysaccharides, midi-GAGR as well as LA-GAGR could protect the neuronsfrom all three neurodegenerative factors, 4HNE, H₂O₂, and Aβ₄₂ peptide.This result indicates that the neuroprotective effect of midi-GAGR doesnot depend on its antioxidant property. To further clarify this, rodentcortical neurons were pre-treated with FGFR1 inhibitor prior to thetreatment with 4HNE. Midi-GAGR could not rescue neurons from4HNE-induced death in neurons pretreated with FGFR1 inhibitor,indicating that FGFR1-mediated neurotrophic signaling pathway is themajor mechanism by which midi-GAGR rescues neurons from oxidative insultinduced death. FGFR1 inhibitor increased the percent of neuron deathcaused by 4HNE to even higher than vehicle plus 4HNE. This indicatesthat FGFR1 is important for neuron survival in the presence of highoxidative insult.

The efficacy of midi-GAGR in enhancing neuronal activity and reducingpathogenic factor in the brains of 3×Tg-AD mice that are known todemonstrate most AD pathogenic progresses until the age of 12 month wasexamined. Consistent with the results of SD rats, intranasallyadministered midi-GAGR increased both NF200 and GAP-43, the markers ofincreased neuritogenesis and synaptic activity, respectively, in thehippocampus. In the cortex, only GAP-43 was increased while NF200 wasnot changed. No change in NF200 in the cortex may be due to thesaturation of the neuritogenic effect of midi-GAGR at the concentrationof equal to or higher than 1 μM, similarly to the result of the in vitroexperiment. In addition to NF200 and GAP-43, PSD95, and pCREB, theindicators of increased synaptic activity, were also increased in bothcortex and hippocampus. The increase in all four markers for enhancedneuronal activity in the brain of 3×Tg-AD mice is strong evidence thatshows a great ability of midi-GAGR for restoring the learning and memoryfunction of the brain in AD mice.

Surprisingly, a reduction of hyperphosphorylated tau (P-Ser202) in thehippocampi of 3×Tg-AD mice was observed. Hyperphosphorylated tau is areliable biomarker for “mild cognitive impairment” (MCI) at early AD.Hyperphosphorylation impairs the function of tau in promotingmicrotubule polymerization, resulting in its aggregation to formneurofibrillary tangles (NFTs), microtubule disassembly, and loss ofaxonal microtubule-based transport. Without wishing to be bound bytheory, it is believed that midi-GAGR may inhibit GSK3β, the majorkinase responsible for tau hyperphosphorylation, in a similar way toFGL.

This Example demonstrates the abilities of a BBB-permeable, long plasmahalf-life, strong neuroprotective and neurotrophic polysaccharide,midi-GAGR. BBB-permeable midi-GAGR can exert its neurotrophic effect inthe cortex and hippocampus for ˜24 h after one-time intranasaladministration. Moreover, midi-GAGR not only increases protein markersfor increased neuronal activity but also reduces hyperphosphorylated tauin the brains of 3×Tg-AD mice. In sum, this Example shows that midi-GAGRhas great therapeutic characteristics (neurotrophic, neuroprotective,BBB-permeable, and long plasma half-life) for the treatment ofneurodegenerative diseases, especially AD.

Example 5 Nesting Behaviors Using Midi-GAGR

3×Tg-AD mice were treated either with sterile water (vehicle) or 0.296nmole of midi-GAGR for 14 days. White feathery papers were scattered at0 hr. After 24-h post treatment, the image of the cages was taken by acamera (FIG. 32A).

The daily sizes (areas) of the nests made by the mice were measuredusing software and used to calculate average nest size (FIG. 32B).

Midi-GAGR-treated 3×Tg-AD mice showed better nesting behavior thanvehicle-treated mice. The nesting behavior is known to be impaired in3×Tg-AD mice, which corresponds to a deterioration of executivefunctions and daily live activities (DLA) in humans. DLA is used alongwith behavioral and psychological symptoms of dementia to diagnose earlyAD in humans. Moreover, nesting behavior heavily depends on thehippocampus. Midi-GAGR rescued some hippocampal function involved innesting behavior.

Example 6 Wound Healing

Midi-GAGR and mini-GAGR were produced from low acyl gellan gum. Low acylgellan gum (an average molecular weight [MW] of ˜99,700 g/mole) wasdigested with α(1→3)-glucosidase for 48 h and 72 h to generate midi-GAGR(4.7 kD) and mini-GAGR (0.8 kD), respectively. The MWs of midi-GAGR andmini-GAGR were determined using a Parallel Plate Rheometer on the basisof the viscosity profile of cleavage products.

Mini-GAGR and midi-GAGR increased blood vessel formation from humanendothelial cells (HUVECs). HUVECs were plated on a matrigel-coated24-well plate (1.2×105/0.3 mL/well), added with Endothelial Cell GrowthMedia Kits (EGM [Lonza, Walkersville, Md.]), and incubated in 5% CO₂ at37° C. for 16-18 h. Then, tubes formed from HUVECs were fixed andlabeled with Acridine Orange (1 μg/ml) and imaged using a fluorescentmicroscope. The total length of tubes per image was measured using‘Metamorph’ software. Treatment with midi-GAGR and mini-GAGR (0.1-1 μM)increased the formation of blood vessels by 2 fold compared to untreatedcontrol (FIG. 33). This result indicates that both midi-GAGR andmini-GAGR have a strong vasculogenic effect. This neovascularizationfacilitates wound healing.

Mini-GAGR and midi-GAGR enhanced the production of GAGs and collagen,which make up the extracellular matrix that fills a wound scar. Humanbone marrow-derived mesenchymal stem cells (hBM-MSCs) were incubated incartilage forming condition plus/minus 0.1-1 μM mini-GAGR or midi-GAGR.As a result, 0.1-1 μM mini-/midi-GAGR drastically enhanced theproduction of type II collagen and glycosaminoglycans (GAGs) incartilage cells (FIGS. 34A-34B). Thus, both midi-GAGR and mini-GAGRenhanced the production of GAGs and collagen, the major tissuecomponents for wound healing.

FGFs are major protein growth factors used for wound healing. GAGRcleavage products bound to an FGF receptor. FIG. 35 shows that mini-GAGRinteracts with FGFR1. Affinity chromatography was performed using epoxysepharose beads to which either dextran (control) or mini-GAGR wasconjugated. After nonspecific binding was blocked and removed by bovineserum albumin and extensive washes, respectively, the beads wereincubated in the plasma membrane extractions of Neuro 2A cells. Thebinding of FGFR1 to the beads was then examined by immunoblotting usinganti-FGFR1 antibody (FIG. 35). It is clear that FGFR1 specifically bindsto mini-GAGR but not to dextran.

Midi-GAGR and mini-GAGR did not enhance adipogenesis. Human BM-MSCs wereincubated in adipogenic media (DMEM containing 4.5 g/L glucose, 10 μg/mLinsulin, 60 μM indomethacin, 10 μM indomethacin, 10% FBS, andantibiotic-antimycotic), and treated with none (control), 0.01, 0.1, or1 μM of midi-GAGR or mini-GAGR every 2nd day for 14 days. Thereafter,cells were stained with Oil-red 0 that stains lipid droplets and imagedusing light microscope. The extents of adipogenesis were measured bymicroplate reader (absorbance at 490 nm). Treatment with midi-GAGR ormini-GAGR did not enhance adipogenesis in MSCs (FIGS. 36A-36B). Thus,these GAGR cleavage products do not cause fat formation.

Midi-GAGR and mini-GAGR do not have a mitogenic effect. Human BM-MSCswere incubated in α-MEM plus 10% FBS and none (control), 0.1, or 1 μM ofmidi-GAGR or mini-GAGR for 7 days. Then, the number of cells was countedusing heamatocytometer (n=6). Neither midi-GAGR nor mini-GAGR increasedthe proliferation of MSCs compared to control, indicating that midi-GAGRand mini-GAGR do not have a mitogenic (tumorigenic) effect.

This Example demonstrates a significant therapeutic ability of midi-GAGRand mini-GAGR for wound healing treatments. Without wishing to be boundby theory, it is believed that low acyl gellan gum has the same effectsas its cleavage products in this regard.

Example 7

Described in this Example is an efficient and effective method to trackan exogenous polysaccharide among endogenous polysaccharides in animals.Instead of using complicated methods and equipment, a fluorescent tag,8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS), can beused to label, track, and quantify a target polysaccharide in animal.75% ethanol can be used to examine the structural intactness ofANTS-polysaccharide in animal sample. TCA and 75% ethanol can be used toseparate free ANTS-polysaccharides from those bound to proteins. Forexample, the following equipment was used: SpectraMax M5 plate reader(Molecular Devices, Sunnyvale, Calif.); VERSA max plate reader(Molecular Devices); SoftMax Pro 5.2. (Molecular Devices);microcentrifuge.

Conjugation of ANTS to Polysaccharide

A 4.7 kD cleavage product of gellan gum (named midi-GAGR: digestion byα(1→3) glycosidase) was tagged with ANTS and examined regarding itsBBB-permeability.

To conjugate ANTS (Molecular Probes, Eugene, Oreg.) to midi-GAGR, 454 of7.4 mM midi-GAGR was mixed with 75 μL of 0.2 M ANTS (7.6 mg ANTS in 8904of acetic acid [3/17, v/v]), which gives the final ratio of 1:400(polysaccharide:ANTS) that is optimal for high-efficiency conjugationbetween polysaccharide and ANTS.

The mixture was briefly vortexed and incubated in an 80° C. water bathfor 30 min. The mixture was then added with 375 μL of 1 M NaCNBH₃(Sigma-Aldrich, St. Louis, Mo.), briefly vortexed, and incubated in an80° C. water bath for 90 mM. Although 37° C. can be also used for 15-hconjugation, 80° C. was used to shorten conjugation time. The mixturewas split into 250-μL aliquots, each of which was mixed with 750 μL of100% pure ethanol to make a final concentration of 75% ethanol.

The mixture was briefly vortexed, incubated at −80° C. for 30 min, andcentrifuged at 3,000×g for 30 min to pellet ANTS-tagged midi-GAGR.

In order to remove free ANTS that might be trapped in ANTS-midi-GAGRpellet, the pellet was washed with 400 μL of 75% ethanol to dissolveANTS trapped in the pellet. After the pellet of ANTS-midi-GAGR wasresuspended in 400 μL of 75% ethanol by pipetting, it wasre-precipitated at 3,000×g for 10 min. This step was repeated threetimes. The final pellet was resuspended in 50 μL of sterile de-ionizedwater.

Centrifugation at 15,700×g was also used for ethanol precipitation ofANTS-tagged polysaccharide. However, it resulted in precipitation offree ANTS. Therefore, the speed of centrifugation was decreased to3,000×g, thus preventing the precipitation of free ANTS while stillprecipitating the similar amount of polysaccharide to that after thecentrifugation at 15,700×g. In another embodiment, 70% ethanol can beused instead of 75% to reduce the precipitation of free ANTS that mightbe trapped in ANTS-polysaccharide pellet. Washes with 70% ethanolprecipitation decreased the amount of ANTS at the pellet; however, therewas also a noticeable loss of polysaccharide at the pellet after eachwash. Thus, 75% ethanol was a desired concentration of ethanol toprecipitate the maximal amount of polysaccharide and the minimal amountof free ANTS.

Calculation of the Conjugation Ratio of ANTS to Polysaccharide inANTS-Polysaccharide

The amounts of ANTS and midi-GAGR in ANTS-polysaccharide conjugate weremeasured by fluorometry and colorimetry, respectively, to calculate theratio of ANTS to midi-GAGR in the conjugate.

For fluorometry, the ANTS-polysaccharide pellet was resuspended anddiluted in 50 of fresh water. The dilutions were placed in the wells ofa 96-well black-wall plate. The emission fluorescence signals(excitation at 350 nm, emission at 520 nm; relative fluorescence units[RFUs]) of the dilutions were measured using SpectraMax M5 plate readerand SoftMax Pro 5.2. A standard curve of ANTS was generated using 0,0.1, 0.3, 1, 3, and 10 mM ANTS to calculate the concentrations of ANTSin the samples.

For colorimetry, a modified phenol-sulfuric acid method modified wasused.

ANTS-polysaccharide pellet was resuspended and diluted in 50 μL of freshwater. The dilutions were placed in the wells of a 96-well clear-wallplate. Each sample was added with 150 μL of concentrated H₂SO₄ and then30 μL of 5% phenol (88% phenol liquefied USP [University of ToledoMedical Center, Toledo, Ohio] diluted in distilled water). The top ofthe plate was covered with a plate sealer and heated at 95° C. for 5min. Using VERSA max plate reader and SoftMax Pro 5.4, the absorbance at490 nm of each well was measured. A standard curve of midi-GAGR wasgenerated using 0, 0.0074, 0.074, 0.74, and 7.4 mM midi-GAGR tocalculate the concentrations of midi-GAGR in the samples.

Calculation of the Ratio of ANTS to Midi-GAGR in the Conjugate

The standard curve of ANTS was generated using the RFUs of 0, 0.1, 0.3,1, 3, and 10 mM free ANTS to quantify the concentrations of ANTS in thesamples. FIG. 37A shows the standard curve of ANTS(R²=0.9975) that wasgenerated on the basis of three different measurements.

The standard curve of midi-GAGR was generated using the absorbances at490 nm for 0, 0.0074, 0.074, 0.74, and 7.4 mM free midi-GAGR. FIG. 37Bshows the standard curve of midi-GAGR (R²=0.9919).

According to the standard curves, the RFU of ANTS-polysaccharide in thepellet before washes was ˜7493 and its absorbance at 490 nm was ˜0.269(FIG. 37C).

After three washes, the mean RFU of ANTS-polysaccharide wassignificantly reduced to ˜4910 while the absorbance at 490 nm was onlyslightly reduced to 0.219. The values of RFU and absorbance at 490 nmwere not further decreased by more washes after the third wash, showingthat most of the loosely-associated free ANTS was removed from thepellet of ANTS-midi-GAGR.

According to the standard curves, the final pellet contained 16.18 mMANTS and 1.55 mM midi-GAGR, which give the ratio of about 10:1 for ANTSto midi-GAGR.

It is to be noted that one ANTS was supposed to be conjugated to onereducing end of a polysaccharide, thus yielding the ratio of 1:1 forANTS to midi-GAGR. However, more ANTS appeared to be conjugated to otherhydroxyl groups on midi-GAGR, yielding the 10:1 ratio for ANTS tomidi-GAGR. Polysaccharide was labeled with ANTS using EDC[1-Ethyl-3-3-dimethylaminopropyl carbodiimide] in order to conjugate theamino group of ANTS to the carboxyl group of glucuronic acid ofmidi-GAGR. However, the EDC conjugate of ANTS-midi-GAGR was notprecipitated by 75% ethanol, showing that the EDC conjugate cannot bepurified using 75% ethanol.)

Measurement of the Amount of ANTS-Polysaccharide that Enters the Brainand Blood Circulation

Administration and Measurement of ANTS-Midi-GAGR

To examine the BBB-permeability of ANTS-midi-GAGR, 40 μL of 1 mMANTS-midi-GAGR was administered into the nostril of Sprague-Dawley rats(male, 350-490 g, age of 8 weeks).

Rats were quickly anesthetized in an isoflurane induction chamber(isoflurane from Henry Schein Animal Health [Dublin, Ohio]). 5%isoflurane is administered into animal by a vaporizer with oxygenflowmeter (0.8-1.5 L/min). The percent of isoflurane was later adjustedto 2% until animal loses righting reflex.

20 μL of 1 mM ANTS-midi-GAGR was intra-nasally administered to eachnostril. Animals were kept in the anesthetized condition for 5 min afterthe administration of ANTS-polysaccharide to prevent the squirting-outof ANTS-polysaccharide from the noses.

At 6 h after the administration of ANTS-polysaccharide, animals weresacrificed using a guillotine. About 1 mL of trunk blood was collectedin a vial immediately after decapitation. Trunk blood was incubated atroom temperature for 1 h to coagulate and centrifuged at 3,000×g for 10min to remove the coagulated, which yields ˜400 μL of serum.Simultaneously, the olfactory bulb tract and whole brain were alsodissected out of the head of the decapitated animal. The brain andolfactory bulb tract were washed with 0.9% saline and homogenized in theequivalent volume of 1×PMEE buffer (pH 7.0; 35 mM KOH, 35 mM PIPES, 5 mMMgSO4, 1 mM EGTA, 1% BSA, and 0.5 mM EDTA) containing 1% Igepal CA-630using a glass homogenizer (Wheaton, Millville, N.J.). The homogenizedbrain extract was centrifuged at 14,500×g for 20 min and at 100,000×gfor 30 min to obtain brain cytosol. The amounts of ANTS-polysaccharidein the serum and the cytosols extracted from olfactory bulb and brainwere measured by fluorometry.

Quantification of ANTS-midi-GAGR in Brain and Blood

The amounts of ANTS-midi-GAGR in the samples were quantified using thestandard curve of ANTS and the conjugation ratio of 10:1 for ANTS tomidi-GAGR (FIGS. 37A-37B).

The RFUs of the cytosols extracted from the olfactory bulb tracts of allrats were lower than ˜50 RFUs that were the same as the basalfluorescence units of those of rats administered with saline alone, thusshowing that little ANTS-midi-GAGR was routed to the olfactory bulbtract.

The RFUs of the sera and brain cytosols were significantly above 50RFUs. The RFU values of brain cytosols and sera were converted to theconcentrations of midi-GAGR using the ratio of 10:1 for ANTS tomidi-GAGR. The brain cytosols and sera of the rats administered withANTS-midi-GAGR contained ˜12 μM (FIG. 20A) and ˜28 μM (FIG. 20B),respectively, of midi-GAGR. Thus, intra-nasally administered midi-GAGRcan enter the brain and blood circulation.

Examination of the Structural Intactness of ANTS-Midi-GAGR in the Serum

It was possible that the fluorescence of the sera was emitted from freeANTS that might be generated by the cleavage of ANTS-midi-GAGR duringthe circulation in the blood. Therefore, it was examined whetherANTS-midi-GAGR in the sera was structurally intact or not by 75% ethanolprecipitation that only precipitate ANTS-midi-GAGR but not free ANTS.

100 μL of the supernatant was added with 300 μL of 100% ethanol to makethe final concentration of 75% ethanol and centrifuged at 3,000×g toprecipitate serum polysaccharides including ANTS-midi-GAGR, leavingpolysaccharide-free ANTS in the supernatant. The pellet was resuspendedin the equivalent volume of water to that of the supernatant. The RFUsof the supernatant and pellet resuspension were measured by fluorometry.The RFUs of the supernatant fell down to below 50 while those of thepellet resuspension were close to those of the supernatant beforeethanol precipitation. This shows that most of ANTS-midi-GAGR wasstructurally intact in the serum.

Examination of the Binding of ANTS-Midi-GAGR to Serum Protein

Also examined was whether ANTS-midi-GAGR in the serum bound to serumproteins like albumin or not. TCA was used to precipitate all theproteins and protein-bound molecules including polysaccharides but leaveprotein-free polysaccharides in the supernatant.

100 μL of serum sample in a microtube was added with 10 μL of TCA tomake the final concentration of 10% TCA, incubated at 4° C. for 10 min,and centrifuged at 15,700×g for 5 min. The supernatant over the pelletwas transferred to a new tube and the pellet containing proteins wasresuspended in 100 μL of water. The RFU of the pellet resuspension wasmeasured by fluorometry. The RFUs of the pellet resuspension was belowthe basal fluorescent units (50 RFUs) while those of the supernatantsstill yielded ˜201 RFUs (FIG. 38C), thus showing that littleANTS-polysaccharide was precipitated along with serum proteins.

100 μL of the supernatant was added with 300 μL of 100% ethanol to makethe final concentration of 75% ethanol and centrifuged at 3,000×g toprecipitate serum polysaccharides including ANTS-midi-GAGR. The pelletwas resuspended in 50 μL water. The RFUs of the supernatant and pelletresuspension were measured by fluorometry. The RFU of the supernatantwas below the basal fluorescent units (50 RFUs) while the pelletresuspension yielded ˜201 RFUs, thus showing that most ANTS-midi-GAGRremained intact in the supernatant after TCA precipitation.

ANTS-midi-GAGR that enters the brain and blood circulation does maintainits intact structure inside animal and does not bind to serum proteinfor 6 h after its intra-nasal administration. Given that thecerebrospinal fluid should contain less digestive enzymes than theperipheral blood, it is now shown herein that ANTS-midi-GAGR in thebrain should be intact as well.)

All publications, including patents and non-patent literature, referredto in this specification are expressly incorporated by reference herein.Citation of the any of the documents recited herein is not intended asan admission that any of the foregoing is pertinent prior art. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicant anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

While the invention has been described with reference to various andpreferred embodiments, it should be understood by those skilled in theart that various changes may be made and equivalents may be substitutedfor elements thereof without departing from the essential scope of theinvention. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from the essential scope thereof. Therefore, it isintended that the invention not be limited to the particular embodimentdisclosed herein contemplated for carrying out this invention, but thatthe invention will include all embodiments falling within the scope ofthe claims.

What is claimed is:
 1. A method of enhancing collagen production incells in a subject in need thereof, the method consisting of:administering an effective amount of a low acyl gellan gum (LA-GAGR)cleavage product to cells as the active ingredient that enhancescollagen production in the cells, and enhancing collagen production inthe cells; wherein the LA-GAGR comprises a polysaccharide based ontetrasaccharide repeating units having a molecular weight ofapproximately 99,600 end and a chemical structure of[D-Glc(β1→4)D-GlcA(β1→4)D-Glc(β1→4)L-Rha(α1→3]n); and wherein theLA-GAGR cleavage product comprises midi-LA-GAGR or mini-LA-GAGR.
 2. Themethod of claim 1, wherein the LA-GAGR cleavage product is produced inthe subject by enzymatic digestion of LA-GAGR by α(1→3)-glucosidase. 3.The method of claim 2, wherein the midi-LA-GAGR has a molecular weightof about 4,775 g/mol.
 4. The method of claim 2, wherein the mini-LA-GAGRhas a molecular weight of about 718 g/mol.
 5. The method of claim 1,wherein the cells comprise cartilage cells or mesenchymal stem cells.